Nanopore biosensors for detection of proteins and nucleic acids

ABSTRACT

Described herein are nanopore biosensors based on a modified cytolysin protein. The nanopore biosensors accommodate macromoiecules including proteins and nucleic acids, and may additionally comprise ligands with selective binding properties.

RELATED APPLICATIONS

This application is a continuation of U.S. Application Ser. No.14/779,895 filed Sep. 24, 2015, which is a national stage filing underU.S.C. § 371 of PCT International Application No. PCT/BE2014/000013,with an international filing date of Mar. 25, 2014, which claims thebenefit under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser.No. 61/805,068, filed Mar. 25, 2013 and which claims foreign prioritybenefits under 35 U.S.C. § 119(a)-(d) or 35 U.S.C. § 356(b) of GBApplication Number 1313477.0, filed Jul. 29, 2013, the contents of eachof which are incorporated herein by reference in their entireties.

FIELD OF INVENTION

The present disclosure relates to nanopore biosensors based on amodified cytolysin protein. The nanopore biosensors accommodate largemolecules including folded proteins and nucleic acids including doublestranded (ds) DNA, and showed augmented activity, solubility andelectrical properties, as compared with other nanopore biosensors.

BACKGROUND

The transport of ions or molecules across a biological membrane is afundamental process in cellular life and is tightly regulated by ionchannels, transporters and pores. Recently, researchers have adoptedbiological,¹ solid-state,² DNA origami³ and hybrid^(3a b 4) nanopores insingle-molecule analysis.⁵ Biological nanopores have advantages comparedto theft synthetic counterparts, mostly because they can be reproduciblyfabricated and modified with an atomic level precision that cannot yetbe matched by artificial nanopores. Biological nanopores, however, alsohave drawbacks. The mechanical stability of biological nanopores dependson individual cases. Alpha hemolysin from Staphylococcus aureus (αHL)and porin A from Mycobacterium smegmatis (MspA) nanopores remain open inlipid bilayers at high-applied potentials and can cope surprisingly wellwith extreme conditions of temperature:⁶ pH^(6b 7) and denaturantconcentrations:^(6b 8) However, most of other porines and channels aremuch less robust. Arguably, however, the biggest disadvantage ofbiological nanopores is their fixed size. For example, the dimensions ofαHL, MspA or aerolysin nanopores allowed the analysis of single strandednucleic acids, aptamers or small peptides,⁹ but their small internaldiameter (˜1 nm) precludes the direct investigations of other importantbiological systems such as folded enzymes or ribozymes.

Recently a significant number of studies sampled the translocation offolded proteins through artificial nanopores.¹⁰ However, theinvestigation of proteins with solid-state nanopores is difficultbecause proteins have a non-uniform charge distribution, they oftenadsorb to the nanopores surface and they translocate too quickly to beproperly sampled.^(10c) Further, because proteins have compact foldedstructure, the diameter of the nanopore should be similar to that of theprotein:^(10b) Recently, we have introduced Cytolysin A from Salmonellatyphi (ClyA) as the first biological nanopore that allows theinvestigation of natively folded proteins.^(7a) The ClyA structure isideal for this task because proteins such as thrombin (37 kDa) or malatedehydrogenase (dimer, 35 kDa monomer) can be electrophoretically trappedbetween the wide cis entrance (5.5 nm, table 1) and the narrower transexit (3.3 nm, table 1), and can therefore be sampled for severalminutes. Ionic currents through ClyA are so sensitive to the vestibuleenvironment that blockades of human and bovine thrombin can be easilydistinguished.^(7a) Our work was based on a ClyA construct where the twonative cysteine residues of ClyA-WT (C87 and C285) were replaced byserine (ClyA-SS),^(7a) However, ClyA-SS monomers showed low watersolubility and low activity when compared to ClyA-WT monomers (FigureS1), and in planar lipid bilayers spontaneously opened and dosed (gated)at applied potentials higher than +60 mV or lower than −90 mV.

Thus, there remains a need in the art for nanopore biosensors with highsensitivity for target analytes as well as high water solubility andstability at a range of potentials. Nanopore biosensors should havefavorable properties of oligomerization, voltage dependent gating, andelectrical noise in single-channel current recordings. The presentdisclosure relates to engineered nanopores in which specificsubstitutions to the native cysteine residues and other residues conferadditional properties as compared with ClyA-WT and ClyA-SS.

SUMMARY

One aspect of the present disclosure relates to a modified ClyA porecomprising a plurality of subunits, wherein each subunit comprises apolypeptide represented by an amino acid sequence at least 80% identicalto SEQ ID NO:1 wherein exactly one Cys residue is substituted with Ser.In some embodiments, the Cys residue is C285. In certain embodiments,each subunit of the modified ClyA pore comprises at least one additionalamino acid substitution selected from L99Q, E103G, F166Y, and K294R. Forexample, each subunit may comprise a polypeptide represented by an aminoacid sequence of SEQ ID NO:2. In certain embodiments, exactly one Cysresidue in each subunit is substituted with Ala. In some embodiments,each subunit of the modified ClyA pore comprises at least one additionalamino acid substitution selected from L203V and H207Y. For example, eachsubunit may comprise a polypeptide represented by an amino acid sequenceof SEQ ID NO:3.

In some embodiments, the modified ClyA pore comprises at least 12subunits. For example, the modified ClyA pore may comprise 12 subunitsor may comprise 13 subunits. In certain embodiments, the modified ClyApore has a cis diameter of at least 3.5 nm. In certain embodiments, themodified ClyA pore has a trans diameter of at least 6 nm. In someembodiments, the modified ClyA pore remains open when the voltage acrossthe modified ClyA pore ranges from −60 +90 to −150mV.

In some embodiments, a protein analyte binds within the lumen of themodified ClyA pore. A protein analyte may bind to more than one sitewithin the lumen of the modified ClyA pore. In some embodiments, theprotein analyte is a protein with a molecular weight in the range of15-70 kDa.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B show a ribbon representation of S. typhi ClyA nanoporesconstructed by homology modeling from the E. coli ClyA structure (PDB:2WCD, 90% sequence identity).¹⁷ In FIG. 1A, one protomer is highlighted,with the secondary structure elements shaded in dark grey from N to Cterminal; other protomers are shown alternating in pale grey. The sidechain of the amino acids changed by directed evolution experiments aredisplayed as spheres. The two native cysteine residues and Phenylalanine166 are labeled. FIG. 1B indicates the sequence changes in ClyA-SS,ClyA-CS and ClyA-AS relative to ClyA-WT.

FIGS. 2A-2B show the oligomerization and nanopore formation of ClyA-WT,ClyA-SS and evolved ClyA variants. FIG. 2A shows the oligomerisation ofClyA nanopores examined by 4-20% BN-PAGE, Proteins (1 mg/ml) werepre-incubated with 0.5% DDM for 30 min at room temperature beforeloading into the gel (40 μg/lane). Lane 1: Protein ladder, lane 2:ClyA-WT, lane 3: ClyA-SS, lane 4: ClyA-AS and lane 5: ClyA-CS. FIG. 2Bshows the unitary nanopore conductance distribution obtained from 100nanopores reconstituted in planar lipid bilayers for ClyA-WT (top),ClyA-CS (middle) and ClyA-AS (bottom) nanopores afterpre-oligomerization in 0.5% DDM. Recordings were carried out at −35 mVin 15 mM Tris.HCl, pH 7.5, 150 mM NaCl and the temperature was 28°C.

FIGS. 3A-3C show the unitary conductance of 62 ClyA-CS nanoporesextracted from the lowest (FIG. 3A), second lowest (FIG. 3B) and thirdlowest (FIG. 3C) oligomeric band of ClyA-CS separated on 4-20%acrylamide BN-PAGE. ClyA-CS monomers were pre-incubated in 0.5% DDM andloaded on a BN-PAGE as described in FIG. 2. The bands that were excisedare boxed and marked with an arrow on the insets. Recordings werecarried out at −35 mV, 28° C. in 15 mM Tris.HCl pH 7.5 containing 150 mMNaCl.

FIGS. 4A-4B show the current blockades provoked by HT on the three typesof ClyA-CS nanopores. In FIG. 4A, from left to right, cut through TypeI, Type II and Type III ClyA nanopores (grey) containing HT (black) inthe nanopore vestibule. Type II and Type III ClyA nanopores were modeledas 13 mer (Type II) or 14 mer (Type III) as described in supplementaryinformation. FIG. 4b shows HT blockades to Type I (left), Type II(middle) or Type III (right) ClyA-CS nanopores at −35 mV. HT currentblockades to Type I and Type II ClyA-CS switched between L1 (IRES%=56±1and 68±1, respectively) and L2 (IRES%=23±3 and 31±1, respectively)current levels. The blockades lasted for several minutes, therefore onlythe first second of the current traces is shown. In Type I ClyA-CS, L1was the most represented current blockade (70%), while in Type IIClyA-CS L2 was mostly populated (96%). HT current blockades to Type IIIClyA-CS nanopores only showed L2 current levels (IRES=32±9). Recordingswere carried out at −35 mV in 15 mM Tris.HCl pH 7.5 150 mM NaCl. Thetraces in FIG. 4B were collected applying a Bessel low-pass filter with2000 Hz cutoff and sampled at 10 kHz and the temperature was 28° C.

FIGS. 5A-5C show protein translocation through Type I and Type IIClyA-CS nanopores. FIG. 5A shows the voltage dependency of HT blockadedwell times determined for Type I (hollow circles) and Type II (filledrectangles) ClyA-CS nanopores. Lifetimes at each voltage were calculatedfrom single exponential fits to cumulative distributions (n≤3)constructed from dwell times of at least 50 blockades. The linesindicate double exponential fits to the experimental points. FIG. 5Bshows the HT current blockades to Type I (left) and Type II (right)ClyA-CS nanopores at −150 mV. The blockades showed only L2 currentlevels for both nanopores (IRES%=23±2 and 31±5, for Type I and Type IIClyA-CS respectively). FIG. 5C shows a typical HT current blockade onType I ClyA-CS at −150 mV showing “shoulder” and “spike” currentsignatures. Recordings were carried in 15 mM Tris.HCl, pH 7.5, 150 mMNaCl in presence of 20 nM HT. The traces in FIG. 5B were collectedapplying a Bessel low-pass filter with 2000 Hz cutoff and sampled at 10kHz. The trace in c was collected applying a Bessel low-pass filter with10 kHz cutoff and sampled at 50 kHz. The temperature was 28° C. Errorsare given as standard deviations.

FIGS. 6A-6D show the characterization of purified ClyA monomers. FIG.6A: Solubility of purified ClyA monomers examined by 4-20% acrylamideBN-PAGE. Equal amounts (40 μg) of purified ClyA monomers (no detergent)were supplemented with ˜10% glycerol and 1× of NativePAGETM RunningBuffer and 1× Cathode Buffer Additive (Invitrogen™) and loaded in eachlane: Lane 1: markers, Lane 2: ClyA-WT, Lane 3: ClyA-SS, Lane 4:ClyA-CS, Lane 5: ClyA-AS. FIG. 6B: Overexpression of ClyA variants.Equal amounts of bacterial pellets derived from overnight culturesoverexpressing ClyA variants were resuspended to ˜100 mg/mLconcentration and disrupted by sonication followed by centrifugation at20′000 g for 10 min (4° C.). 20 μL of the supernatant containing thesoluble fraction of ClyA proteins were loaded on lanes 2, 4, 6 and 8 ofa 12% acrylamide SDS-PAGE. The lysate pellets were brought to theoriginal volume by adding a solution containing 15 mM, Tris.HCl pH 7.5,150 mM NaCl and 2% SDS w/v. 20 μL of such solution were loaded on lanes3, 5, 7 and 9 of the same 12% acrylamide SDS-PAGE. Therefore, Lane 1:protein marker, Lanes 2 and 3: ClyA-SS supernatant and pellet fractions,respectively; Lanes 4 and 5: ClyA-WT supernatant and pellet fractions,respectively; Lane 6 and 7: ClyA-CS supernatant and pellet fractions,respectively; and Lane 8 and 9: ClyA-AS supernatant and pelletfractions, respectively. FIG. 6C: Hemolytic assays. ClyA monomers (0.6μM) were incubated with 100 μL of 1% horse erythrocytes suspension (110μL final volume) and the decrease of turbidity was measured at 650 nm(OD650 nm). ClyA-WT is shown as a thick grey line, ClyA-SS as a thickblack line, ClyA-CS as a thin black line and ClyA-AS as a dashed line.FIG. 6D: The rates of hemolysis (calculated as the inverse of the timeto reach 50% of turbidity) plotted against protein concentration ClyA-WT(triangle, thick grey line), ClyA-SS (squares, thick black ClyA-AS(circles, dashed line) and ClyA-CS (diamonds, thin black line).

FIGS. 7A-7B show an example of the screening of the oligomerization ofClyA variants using 4-20% acrylamide BN-PAGE. FIG. 7A: Round 4. Lane 1,2: ClyA-CS, lane 3 and 4: 4ClyA5, lane 5 and 6: 4ClyA3, lane 7 and 8:4ClyA1, lane 9 and 10: 4ClyA2, lane 11 and 12: 4ClyA6. Samples wereprepared as explained in FIG. 6A supplemented with 0.05% (even lanenumber) or 0.1% (odd lane numbers) SDS. SDS was used to counter the“smearing” effect of large quantity of DDM in the samples. FIG. 7B:Round 5. Lane 1, 2: 5ClyA2, lane 3: 5ClyA1, lane 4: ClyA-AS. Sampleswere supplemented with 0.05% SDS. Oligomerization was triggered by theaddition of 1% DDM and ClyA variants were partly purified by Ni-NTAaffinity chromatography as described in methods.

FIG. 8 shows noise characteristics of three types of ClyA nanoporesunder −35 mV potential in 150 mM NaCl, 15 mM Tris.HCl. Current powerspectral densities of the Type I (dashed line), Type II (dotted line)and Type III (light gray solid line) ClyA-CS nanopores at −35 mVobtained from 0.5 s traces. The current power spectral density at 0 mVis shown in black. Each line corresponds to the average of power spectracalculated from 3 recordings carried out on different single channels.

FIGS. 9A-9B show the duration of HT (FIG. 9A) and FP (FIG. 9B) blockadeson Type I ClyA-SS pores. Traces were recorded at a sampling rate of 10kHz with an internal low-pass Bessel filter set at 2 kHz. Each plottedvalue corresponds to the average determined using at least 3 differentsingle channels. Errors are given as standard deviations.

FIG. 10 shows the typical HT current blockades on Type I ClyA at −150 mVshowing “shoulder” and “spike” current signatures. Recordings werecarried in 15 mM Tris.HCl, pH 7.5, 150 mM NaCl in presence of 20 nM HT.The traces were filtered with a Gaussian low-pass filter with 10,000 Hzcutoff filter and sampled at 50,000 Hz.

FIGS. 11A-11D show dsDNA translocation through ClyA nanopores. On theright of the current traces, ClyA (pore shaped structure) andneutravidin (dark gray) are depicted, and the dsDNA is shown as a blackline. FIG. 11A: Sections through of ClyA from S. typhi constructed byhomology modeling from the E. coli ClyA structure (PDB: 2WCD, 90%sequence identity),²² ClyA is shown with pore measurements including theVan der Waals radii of the atoms, and the approximate location ofresidue 103 (serine in the WT sequence) on the pore indicated. FIG. 11B:at +100 mV (level I_(O+100)=1.74±10.05 nA), the addition of 0.12 μM of290 bp dsDNA 1 to the cis side of a ClyA nanopore provokes short currentblockades of I_(RES)=0.63±0.01 (level 1*₊₁₀₀=1.10±0.03 nA, n=3) that aredue to the translocation of dsDNA through the pore. Addition ofneutravidin to the cis chamber converted the short current blockades tohigher conductive and long-lasting current blockades withI_(RES)=0.68±0.01 (level 1₊₁₀₀=1.19±0.01) due to the partialtranslocation of DNA through the pore. The open pore current wasrestored by reversing the potential to −100 mV (red asterisk). The insetshows a typical transient current blockade. FIG. 11C: Details for acurrent blockade due to the formation of a cis pseudorotaxanes at +100mV. FIG. 11D: Formation of a trans pseudorotaxanes at −100 mV bythreading the dsDNA:neutravidin complex (see above) from the trans side(level 1⁻¹⁰⁰=1.02±0.03 nA, I_(RES)=0.62±0.01, n=4). The electricalrecordings were carried out in 2.5 M NaCl, 15 mM Tris.HCl pH 7.5 at 22°C. Data were recorded by applying a 10 kHz low-pass Bessel filter andusing a 20 μs (50 kHz) sampling rate.

FIGS. 12A-12E show formation of a nanopore-DNA rotaxane. FIG. 12A:representation of the hybridisation of the DNA molecules used to formthe rotaxane. Arrowheads mark the 3′ ends of strands. FIG. 12B: rotaxaneformation. At −100 mV following the addition of the DNA hybrid 3 (1 μM)complexed with neutravidin (0.3 μM) and oligo 4 (1 μM) to the transcompartment, the open pore current of ClyA-2 (I_(O−100)=4.71±0.07 nA,n=4) is reduced to level I⁻¹⁰⁰=1.1±0.04 nA (I_(RES) value of 0.64±0.02,n=4), indicating that dsDNA threads the pore from the trans side.Stepping to +100 mV (red asterisk) produced a current block withI_(RES)=0.77±0.04 (level 2₊₁₀₀=1.31±0.09 nA, n=4), indicating that theDNA is still occupying the pore at positive applied potentials. Level 2most likely corresponds to ssDNA occupying the vestibule of the pore.Successive switching to positive applied potentials did not restore theopen pore current (FIG. 16), confirming that a rotaxane is permanentlyformed. FIGS. 12C-12E: Rotaxane removal. FIG. 12C: at +100 mV the ioniccurrent through ClyA-2 nanopores showed a multitude of fast currentblockades (FIG. 15), suggesting that the ssDNA molecules attached at thecis entrance of the pore transiently occupy the lumen of the pore. FIG.12 d: after the rotaxane is formed (FIG. 12B), at +100 mV the nanoporeshows a steady ionic current (level 2₊₁₀₀) suggesting that a single DNAmolecule is occupying the pore. FIG. 12E: 20 minutes after the additionof 20 mM DTT to the cis compartment the DNA molecules atop the ClyA poreare removed and the open pore current at +100 mV is restored(I_(O+100)=1.78±0.07, n=4). Recordings were performed as described inFIG. 11.

FIG. 13 shows the ssDNA blockades to ClyA-CS. At +100 mV, the additionof 2 μM of a biotinylated ssDNA (5a) to the cis side of ClyA-CSnanopores in the presence of 0.6 μM neutravidin provoked transientcurrent blocks (1.24±0.02 nA, I_(RES)=0.69±0.04, n=3), indicating thatssDNA can enter the lumen of the pore but only transiently. Thesubsequent addition of the complementary ssDNA strand (5b) converted thecurrent blockades into level 1₊₁₀₀ blocks (1.22±0.13 nA,1_(RES)=0.67±0.01, n=3), indicating that the dsDNA can now translocatethe entire length of the pore.

FIGS. 14A-14D show the transport of DNA through ClyA nanopores. FIG.14A: schematic representation of the strand displacement reaction thatpromotes the release of DNA from the pore. FIG. 14B: at +50 mV and inthe presence of 3 (0.3 μm) ClyA-2 showed a steady open pore current(I_(O+50)=0.85±0.01 nA, n=3), showing that the ssDNA strands attached tothe pore do not thread through the lumen of the pore and prevent thetranslocation of dsDNA form solution. FIG. 14C: the addition of thessDNA strands 6 (40 nM) to the cis chamber produced long lasting currentblockades with I_(RES)=0.70±0.02, (level 2₊₅₀=0.59±0.02 nA, n=5)indicating that the dsDNA hybrid is threaded the pore. FIG. 14D: thesubsequent addition of 1 μM of 7 to the trans chamber (+50 mV), whichalso contains 0.3 μM neutravidin, promoted the release of the DNA threadand restored the open pore current. Subsequently, dsDNA molecules aresequentially captured and released as shown by multiple blocked and openpore currents. For the sake of clarity, neutravidin is not included inthe cartoon representation. The electrical recordings were carried outin 2.5 M NaCl, 15 mM Tris.HCl pH 7.5 at 22° C. Data were recorded byapplying a 10 kHz low-pass Bessel filter and using a 20 μs (50 kHz)sampling rate. The current signal in panel d was digitally filtered at 2kHz with a post-acquisition low-pass Gaussian filter. The appliedpotential of this experiment was set to +50 mV to facilitate theobservation of the multiple block and release (FIG. 14D), as at higherapplied potentials the capture of the DNA in cis is very fast.

FIGS. 15A-15D show the results of control experiments showing that allcomponents of FIG. 12 are necessary to form a DNA rotaxane. FIG. 15A:the absence of the bridging sequence 4 does not allow the linkagebetween ClyA-2 and 3. After the complex is captured at −100 mV it isreadily expelled from the pore at +100 mV (asterisk) as shown by thetypical current signature of an open pore current for ClyA-2 at +100 mV.FIG. 15B: the absence of 2 from the pore top (e.g. after cleavage withDTT) does not allow 3·4 DNA hybrid to bind to the pore when captured at−100 mV. Upon reversing the potential to +100 mV (red asterisk) the openpore current is restored. FIG. 15C: Typical current recording for aClyA-2 nanopore at +100 mV. FIG. 15D: all points histogram (5 pA binsize) for 20 seconds of the current trace shown in FIG. 15A. LevelI_(O+100)=1.71±0.07 nA, level A=1.62±0.12 nA, level B=1.43±0.05 nA andlevel C=1.28±0.06 nA, corresponding to the I_(RES) values of 0.94±0.04,0.84±0.03 and 0.75±0.03, respectively. The values, calculated from 12experiments, might represent the DNA strands lodging within the lumen ofClyA.

FIGS. 16A-16B show the Current versus voltage (IV) relationships forClyA-2 nanopores. FIG. 16A: a typical curve for ClyA-2 before (lightestgrey line) and after (black line) rotaxane formation. The medium-greyline indicates the same nanopore after the DNA molecules attached to thenanopore are removed by the addition of DTT. The current recordings weremeasured by applying an automated protocol that ramped the voltage from−100mV to +100 mV in 4 seconds. FIG. 16B: I-V curves calculated from theaverage of four experiments showing the steady-state (1 s) ClyA-2 openpore current levels (white spheres) and ClyA-2 open pore current levelsin a rotaxane configuration (back spheres). The unitary conductancevalues of the nanopores as calculated from the slopes of the I-V curveswere 17.1 nS for ClyA-2 at both positive and negative bias, 10.8 nS forthe rotaxane at negative bias and 13.0 nS at positive bias. Therotaxanes were prepared as described in FIG. 12.

FIGS. 17A-17F show dsDNA current blockades to ClyA-CS. On the right ofeach current trace the cartoon represents the physical interpretation ofthe current recordings, FIGS. 17A-17D: current recordings for ClyA-2after hybridisation with a 6·7 (indicated with asterik) at differentapplied potentials. FIG. 17E: at +50 mV, upon hybridisation with 6 (40nM) and 2, the DNA duplex 3 (0.3 μM) is transported through the pore asshown by the drop in the ionic current from I_(O+50)=0.85±0.01 nA, to alevel 2₊₅₀ block (I_(RES)=0.70±0.02). Reversal of the applied potentialto −50 mV restores the open pore current (O_(O−50)=0.83±0.00). FIG. 17F:the subsequent addition of 1 μM neutravidin (black) to the trans chamberlocked the DNA thread within the pore as revealed after the reversal ofthe potential to −50 mV when a blocked pore level (I_(RES)=0.67±0.02)was observed.

FIGS. 18A-18C show selective DNA translocation through ClyA-2 pores.FIG. 18A: left, at +50 mV the ionic current through ClyA-2 nanoporesshowed fast and shallow current blockades, suggesting that the ssDNAmolecules attached at the cis entrance might transiently occupy thelumen of the pore. Middle, after dsDNA strand 1 (50 nM) is added to thecis chamber the current signals did not change, indicating that dsDNAdoes not translocate ClyA-2. Right, 20 minutes after the addition of 20mM DTT to the cis compartment the DNA molecules atop the ClyA pore areremoved and the DNA can translocate through the pore. FIG. 18B: sameexperiment as described in panel a but in the presence of 1 μM ofneutravidin. FIG. 18C: same as in FIG. 18B, but at +100 mV.

FIGS. 19A-19C show Voltage dependence of the interaction of assDNA-dsDNA hybrid construct with ClyA-CS pores. FIG. 19A: depiction ofthe DNA molecules used in the experiments described in FIG. 19B and FIG.19C. FIG. 19B: current blockades of the DNA hybrid 3 (FIG. 19A, left) incomplex with neutravidin at +50 mV (left), +70 mV (middle) and +100 mV(right). FIG. 19C: current blockades of the DNA hybrid 3:6 (FIG. 19A,right) in complex with neutravidin at +50 mV (left), +70 mV (middle) and+100 mV (right).

FIG. 20 shows strand release with Boolean logic. The OR gate isrepresented at the top and the AND gate at the bottom. DNA isrepresented as directional lines, with the arrow head denoting the 3′end. The sections within the same DNA strand represent DNA domains thatact as a unit in hybridization, branch migration or dissociation.Domains are represented by the letter followed by a number. T denotes atoehold domain. A starred domain represents a domain complementary insequence to the domain without a star. FIG. 14 shows the implementationof the first reaction of the OR gate.

FIGS. 21A-21D show alternative transport of DNA through ClyA nanopores.FIG. 21A: schematic of the release of the DNA thread by stranddisplacement showing the implementation of the second reaction of the ORgate described in FIG. 18. FIG. 21B: open pore current of ClyA-2 at +35mV. FIG. 21C: addition of DNA strands 3 (1 μM) and 8 (0.5 μM) to the cischamber produce long lasting current blockades, indicating that thedsDNA hybrid is threading the pore, FIG. 21D: at +35 mV the subsequentaddition of 1 μM of 9 to the trans chamber, which also might contain 1μM neutravidin, promoted the release of the DNA thread and restored theopen pore current. Subsequently, other dsDNA molecules are captured andthen released as shown by the cycles of blocked and open pore currents.Data were recorded by applying a 10 kHz low-pass Bessel filter and usinga 20 μs (50 kHz) sampling rate.

DETAILED DESCRIPTION

The present disclosure relates to nanopore biosensors which may be usedfor a variety of applications, including but not limited to detectionand quantification of proteins and translocation of DNA. The nanoporesare based on pore-forming bacterial cytotoxins, which act as channelsfor large macromolecules such as proteins and nucleic acids. Nucleicacids may include DNA, for example, single stranded DNA (ssDNA) ordouble stranded DNA (dsDNA), RNA, peptide nucleic acids, and/or nucleicacid analogs.

Modified Nanopore Biosensors

One aspect of the present disclosure relates to nanopore biosensors madefrom modified pore proteins. Exemplary pore proteins include, but arenot limited to cytolysins, hemolysins, porins, DNA packaging protein,and motor proteins. Pore proteins may be bacterial or viral proteins.

In certain embodiments, the modified pore protein is a pore-formingcytotoxic protein, for example, a bacterial cytolysin. The cytolysin maybe a Cytolysin A (ClyA) from a gram-negative bacteria such as Salmonellaor Escherichia coli (E. coli). In some embodiments, the modifiedcytolysin is a modified ClyA from Salmonella typhi (S. typhi) orSalmonella paratyphi (S. paratyphi). In some embodiments, the modifiedClyA pore comprises a plurality of subunits, wherein each subunitcomprises a polypeptide represented by an amino acid sequence at least80% identical to SEQ ID NO: 1. In certain embodiments, the subunits arerepresented by an amino acid sequence at least 85% identical, 90%identical, 95% identical, or 100% identical to SEQ ID NO:1, identicalmay refer to amino acid identity, or may refer to structural and/orfunctional identity. Accordingly, one or more amino acids may besubstituted, deleted, and/or added, as compared with SEQ ID NO:1.Modifications may after the pore lumen in order to after the size,binding properties, and/or structure of the pore. Modifications may alsoafter the ClyA pore outside of the lumen.

In certain embodiments, each subunit comprises a polypeptide representedby an amino acid sequence at least 80% identical to SEQ ID NO:1, whereinexactly one Cys residue is substituted with Ser. Each subunit may berepresented by an amino acid sequence that is at least 85%, 90%, 95%,96%, 96%, 98%, or 99% identical to SEQ ID NO:1, and additionally exactlyone Cys residue may be substituted with Ser. The Cys residue may be Cys87 and/or Cys 285 in SEQ ID NO:1. In some embodiments, the Cys residueis Cys285. Other amino acid residues may be substituted, for example,with amino acids that share similar properties such as structure,charge, hydrophobicity, or hydrophilicity. In certain embodiments,substituted residues are one or more of L99, E103, F166, and K294. Forexample, the substituted residues may be one or more of L99Q, E103G,F166Y, and K294R. An exemplary subunit may comprise substitutions L99Q,E103G, F166Y, K294R, and C285S. Thus, each subunit may comprise apolypeptide represented by an amino acid sequence of SEQ ID NO:2. Anexemplary modified ClyA pore comprising subunits in which exactly oneCys residue is substituted with Ser may be called ClyA-CS.

The modified ClyA pore may comprise a plurality of subunits, whereineach subunit comprises a polypeptide represented by an amino acidsequence that is at least 80% identical to SEQ ID NO:1, wherein exactlyone Cys residue is substituted with Ala. The cysteine residue may be Cys87 or Cys 285. Each subunit may be represented by an amino acid sequencethat is at least 85%, 90%, 95%, 96%, 96%, 98%, or 99% identical to SEQID NO:1, and exactly one Cys residue may be substituted with Ser and/orexactly one Cys residue may be subsituted with Ala. In some embodiments,each subunit is represented by an amino acid sequence that is at least85%, 90%, 95%, 96%, 96%, 98%, or 99% identical to SEQ ID NO:1, andadditionally exactly one Cys residue may be substituted with Ser andexactly one Cys residue may be subsituted with Ala. Other amino acidresidues may be substituted, for example, with amino acids that sharesimilar properties such as structure, charge, hydrophobicity, orhydrophilicity. In certain embodiments, substituted residues are one ormore of L99, E103, F166, K294, L203 and H207. For example, thesubstituted residues may be L99Q, E103G, F166Y, K294R, L203V, and H207Y.An exemplary subunit may comprise L99Q, E103G, F166Y, K294R, L203V, andH207Y, and C285S. Accordingly, each subunit may comprise a polypeptiderepresented by an amino acid sequence of SEQ ID NO:3. An exemplarymodified ClyA pore comprising subunits in which exactly one Cys residueis substituted with Ser and exactly one Cys residue is substituted withAla may be called ClyA-AS.

The present disclosure further relates to nucleic acids encoding themodified ClyA pores. In some embodiments, a nucleic acid encoding amodified ClyA pore is represented by a nucleotide sequence that is atleast 80%, 90%, 95%, 96%, 96%, 98%, or 99% identical to SEQ ID NO:4. Anucleic acid may be represented by SEQ ID NO: 5 or SEQ ID NO:6.Nucleotide sequences may be radon optimized for expression in suitablehosts, for example, E. coli.

The modified ClyA pore may have a pore lumen of at least 3 nm indiameter, for example, the diameter may measure 3 nm, 3.5 nm, 4 nm, 4.5nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, or greater. The size of the porelumen may depend on the analyte to be detected by the modified ClyApore. The cis diameter of the pore lumen may be at least 3.5 nm and/orthe trans diameter of the ClyA pore may be at least 6 nm. In general,cis refers to the end of the modified ClyA pore to which an analyte isadded, while trans refers to the end of the modified ClyA pore throughwhich the analyte exits after translocating the length of the porelumen. In artificial lipid bilayers, for example, the trans end of apore may be inserted in the lipid bilayer, while the cis end of the poreremains on the same side of the lipid bilayer. Accordingly, the cisdiameter of the pore is the diameter of the opening at the cis end ofthe pore, and the opening to which an analyte is added, while the transdiameter of the pore is the diameter at the opening of the trans end ofthe pore, from which an analyte exits.

The size of the pore lumen may also depend on the number of subunits inthe modified ClyA pore. For example, larger pores which are made up of13 or 14 subunits may have larger lumens than pores made up of 7subunits. In some embodiments, the modified ClyA pores comprise 12 ormore subunits. In certain embodiments, the modified ClyA pores comprise12 subunits. In certain embodiments, the modified ClyA pores comprise 13subunits, or comprise 14 subunits. The subunits may preferentiallyassemble in 12 mers and/or tamers, depending on the amino acid sequenceof the subunits. In some embodiments, each subunit comprises apolypeptide as disclosed herein. Within a single modified ClyA pore,each of the subunits may be identical, or the subunits may be different,so that subunits in a modified ClyA pore may comprise sequences thatdiffer from sequences of other polypeptide subunits in the same modifiedClyA pore. In certain embodiments, modified ClyA pores as disclosedherein, such as ClyA-CS pores, may form more than one subtype dependingon subunit composition. For example, there may be at least 2 or 3different subtypes (i.e., Type I, Type II, Type III) of modified ClyA-CSpore, depending on subunits. Each subtype may have different conductancemeasurements, as compared with other subtypes. Subtypes may bepreferentially formed by subunits of a particular polypeptide sequence.

The substitutions in specific residues may confer new properties on themodified ClyA pores, as compared with wild-type ClyA pores found innature. Voltage dependent opening and dosing (gating) of the pore atspecific voltages is one property. In planar lipid bilayers, forexample, ClyA-SS spontaneously opens and closes at applied potentialsthat are greater than +60 mV or lower than −90 mV. In some embodiments,the modified ClyA pores as described herein remain open when the voltageacross the pore (i.e., the voltage across the membrane which themodified ClyA pore is in) ranges from +90 mV to −150 mV. Accordingly,the modified ClyA pore may remain open when the voltage across the poreis held at +90, +85, +80, +75, +70, +65, +60, +55, +50, +45, +40, +35,+30, +25, +20, +15, +10, +5, 0, −5, −10, −15, −20, −25, −30, −35, −40,−45, −50, −60, −65, −70, −75, −80, −85, −90, −95, −100, −110, −115,−120, −125, −130, −135, −140, −145, −150 mV, and/or the voltage acrossthe pore is adjusted between +90 mV and −150 mV (inclusive), or anysubrange of voltages in between. In certain embodiments, the modifiedClyA pores show low electrical noise as compared with the signal (i.e.,the current block measured). Thus, the noise inherent in a modified ClyApore is reduced when pores as described herein are used. An exemplarymodified ClyA pore shows noise measurements of ______1.5 pA rms______to3 pA rms______under______−35 mV in 150 mM NaCl 15 mM Tris.HCl pH7.5______conditions. Notably, it is possible to reduce noise byincreasing the salt concentration and/or altering the length of timeduring which a current block is measured.

In some embodiments, the modified ClyA pores show solubility propertiesthat differ from wild-type ClyA pores. For examples, monomers of themodified ClyA pores may be soluble in water, and/or in other solutionswhere surfactants such as SDS or DDM are not present. Stable oligomersare modified ClyA pores that are capable of withstanding appliedpotentials of +150 mV to −150 mV across membranes or lipid bilayers intowhich the modified ClyA pores are inserted.

Nanopores with Ligands

A further aspect of the present disclosure relates to nanoporebiosensors in which modified

ClyA pore proteins are combined with ligands that have selective bindingproperties. In some embodiments, these modified pores and ligands areused to identify protein analytes in complex biological samples, forexample, in a tissue and/or a bodily fluid. The target protein analytemay be present in a low concentration as compared to other components ofthe sample. In some embodiments, ligands may also be used to targetsubpopulations of macromolecular analytes based on conformation or onfunctional properties of the analytes. The presence of a ligand mayincrease the association of the target protein analyte with the modifiedpore. For example, the ligands may act as a selectivity filter at theentrance of the pore, increasing capture of the target protein whilerepulsing other non-target proteins in the sample.

Exemplary ligands include but are not limited to aptamers, antibodies,receptors, and/or peptides that bind to the target protein. In someembodiments, ligands may be inhibitors of the target protein, whichsuppress the binding of the target protein to the modified ClyA pore.

In certain embodiments, a ligand that binds to a target protein analyteis added to a sample prior to the detection steps described above. Thisstep may provide additional confirmation that a target protein analyteis present. Thus, a method for detecting at least one target protein ina sample may comprise comprises (a) contacting the sample with a ligandthat binds to a target protein; (b) contacting the sample with amodified ClyA pore as disclosed herein; (b) applying an electricalpotential across the modified ClyA pore; (c) measuring electricalcurrent passing through the modified ClyA pore at one or more timeintervals; and (d) comparing the electrical current measured at one ormore time intervals with a reference electrical current, wherein achange in electrical current relative to the reference current indicatesthat the presence of the target protein in the sample. In addition, thechange in electrical current may be compared with a sample that was notcontacted with a ligand prior to measuring the electrical currentthrough the modified ClyA pore. If a target protein analyte is indeedpresent, the addition of a ligand will suppress the binding of thetarget protein to the modified ClyA pore, and a current block would notbe detected. In contrast, a current block would be detected when theligand was not added. With both results together, the presence and theconcentration of the target protein could be determined. For example, ina given sample containing many different proteins including a targetprotein analyte, the sample may initially give X blockades per second.After addition of an excess of a specific ligand, the sample may give(X-n) blockades per second. Thus, n may reflect the blockades per secondproduced by the target protein analyte in the original sample, which, inturn, may provide information about the concentration of the targetprotein analyte in the original sample.

In certain embodiments, varying electrical potentials are applied acrossthe modified ClyA pore. For example, the electrical potential appliedacross the modified ClyA pore may range from −90 mV to +90 mV. Theelectrical potential may be −90 mV, −85 mV, −80 mV, −75 mV, −70 mV, −65mV, −60 mV, −55 mV, −50 mV, −45 mV, −40 mV, −35 mV, −30 mV, −25 mV, −20mV, −15 mV, −10 mV, −5 mV, 0 mV, +5 mV, +10 mV, +15 mV, +20 mV, +25 mV,+30 mV, +35 mV, +40 mV, +45 mV, +50mV, +55 mV, +60 mV, +65 mV, +70 mV,+80 mV, +85 mV, and/or +90 mV. For each potential, the electricalcurrent may be measured and compared with one or more referenceelectrical currents. In some embodiments, a reference electrical currentis measured in an open, unblocked pore. In some embodiments, a referenceelectrical current is measured in a modified ClyA pore that is bound toa known protein, for example, a protein whose presence or absence willbe determined in solution.

In some embodiments, a modified ClyA pore as described herein may beconjugated to one or more aptamers. When more than one aptamer isconjugated, the aptamers may be the same aptamer or may be differentaptamers. The one or more aptamers may be conjugated to a cysteineresidue in the modified ClyA pore. In some embodiments, a modified ClyApore comprises a cysteine residue in pace of another amino acid residuein the pore. This cysteine residue substitution may be combined withother amino acid substitutions, deletions, and/or additions maderelative to the wild-type pore protein. For example, modificationswithin the pore lumen may be engineered to after the size, bindingproperties, and/or structure of the pore. In some embodiments, cysteineresidues are substitued with other amino acids such as serine residues.The modified pore protein may be a pore comprising multiple subunits,for example, 12 subunits, in which at least one subunit comprises amodified amino acid. In certain embodiments, the modified ClyA is from agram-negative bacteria such as Salmonella or Escherichia coli (E. coli).In some embodiments, the modified cytolysin is a modified ClyA fromSalmonella typhi (S. typhi) or Salmonella paratyphi (S. paratyphi). Insome embodiments, the modified A modified ClyA pore comprises 12subunits, each subunit comprising a sequence shown in SEQ ID NO: 2.

An aptamer may be a nucleic acid aptamer comprising DNA, RNA, and/ornucleic acid analogs. An aptamer may be a peptide aptamer, such as apeptide aptamer that comprises a variable peptide loop attached at bothends to a scaffold. Aptamers may be selected to bind to a specifictarget protein analyte. In certain embodiments, two or more aptamers areconjugated to the same modified ClyA pore. For example, 5, 6, 7, 8, or 9aptamers may be conjugated to a modified ClyA pore. Alternatively, 10,11, or 12 aptamers may be conjugated to a modified ClyA pore. If morethan one aptamer is conjugated to the modified ClyA pore, the aptamersmay be positioned at least 2 nm apart.

In certain embodiments, a modified ClyA pore as described herein iscombined with one or more peptide ligands. Peptide ligands may beattached to the modified ClyA pores via disulfide linkages,cross-linking, and/or chemical ligation. The modified ClyA pore may alsobe engineered as a fusion protein in which one or more peptide ligandsis fused to at least one subunit of the modified ClyA pore. In someembodiments, the modified ClyA pore is combined more than one peptideligand. The peptide ligands may be the same ligand or may be differentligands. Exemplary peptide ligands include, but are not limited to,receptors, antibodies, inhibitors, activators, and/or other peptideligands that bind to target proteins.

Target Analytes

1. Protein Analytes

In some embodiments, the modified ClyA pore protein is engineered toallow protein analytes to bind within the lumen of the pore. Thisbinding mediates a robust, reproducible current block, which is readilydistinguished from the unblocked ionic currents measured in unboundpores. The protein analytes may range from 15-70 kDa in molecularweight, for example, exemplary protein analytes may have a molecularweight of 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 kDa. Theanalyte may be a dimer or other multimer of a smaller protein.

In some embodiments, the modified ClyA pore proteins comprise more thanone site of binding and/or residence for protein analytes. For example,a modified ClyA pore protein may comprise two sites: level 2 may beassociated with residence of the protein analyte at a deep, moresterically constrained site, while the level 1 may be associated withresidence of the protein analyte at a position closer to the wider cisentrance of the pore. Protein analyes may move between the two sites,thus eliciting two current levels seen within the same current blockadeevent. Thus, a modified ClyA pore proteins may provide more than one(Le., two) current level measurement upon binding to a protein analyte.

In some embodiments, the modified ClyA-CS pores as described herein arecapable of detecting and quantifying protein analytes. The modified ClyApores may distinguish between homologs of the same protein, for example,bovine thrombin and human thrombin.

a. Detection and Identification of Proteins

Another aspect of the present disclosure relates to detection ofspecific proteins in a sample. In some embodiments, a method fordetecting the presence of at least one protein analyte in a samplecomprises (a) contacting the sample with a modified ClyA pore asdisclosed herein; (b) applying one or more electrical potentials acrossthe modified ClyA pore; (c) measuring current passing through themodified ClyA pore at each of the one or more electrical potentials; and(d) comparing measured currents with reference currents, wherein achange in currents relative to the reference currents indicates thepresence of the protein analyte in the sample. In some embodiments, thechange in currents is a decrease in current. In some embodiments, atarget protein has a molecular weight in the range of 15-50 kDa, forexample, a molecular weight of 15, 20, 25, 30, 35, 40, 45, or 50 kDa.The reference current may be a current measured through the modifiedClyA pore in the absence of a ligand, and/or a current measured throughthe modified ClyA pore in the presence of a reference ligand. In someembodiments, the reference ligand and the protein analyte are identical.In certain embodiments, the reference ligand and the protein analyte areat least 75%, 80%, 85%, 90%, or 95% identical. Thus, in someembodiments, a method for identifying a protein analyte in a samplecomprises (a) contacting the sample with a modified ClyA pore asdescribed herein; (b) applying one or more electrical potentials acrossthe modified ClyA pore; (c) measuring currents passing through themodified ClyA pore at each of the one or more electrical potentials; and(d) comparing measured currents with one or more reference currents froma known ligand, wherein a match between the measured currents and thereference currents indicates the protein analyte and the known ligandare identical. Similarly, a non-match between the measured currents andthe reference currents may indicate that the protein analyte and theknown ligand are not identical.

In some embodiments, the modified ClyA pore comprises amino acidsubstitutions, deletions, and/or additions, as compared with thewild-type pore protein. For example, modifications within the pore lumenmay be engineered to after the size, binding properties, and/orstructure of the pore. In some embodiments, cysteine residues aresubstitued with other amino acids such as serine residues. The modifiedpore protein may be a pore comprising multiple subunits, for example,between 7-11 subunits, 12 subunits, 13 subunits, or 14 subunits, inwhich at least one subunit comprises a modified amino acid. In certainembodiments, the modified ClyA is from a gram-negative bacteria such asSalmonella or Escherichia coli (E. coli). In some embodiments, themodified cytolysin is a modified ClyA from Salmonella typhi (S. typhi)or Salmonella paratyphi (S. paratyphi). In some embodiments, themodified A modified ClyA pore comprises 12 subunits, each subunitcomprising a sequence shown in SEQ ID NO: 1.

In certain embodiments, varying electrical potentials are applied acrossthe modified ClyA pore. For example, the electrical potential appliedacross the modified ClyA pore may range from −90 mV to +90 mV. Theelectrical potential may be −90 mV, −85 mV, −80 mV, −75 mV, −70 mV, −65mV, −60 mV, −55 mV, −50 mV, −45 mV, −40 mV, −35 mV, −30 mV, −25 mV, −20mV, −15 mV, −10 mV, −5 mV, 0 mV, +5 mV, +10 mV, +15 mV, +20 mV, +25 mV,+30 mV, +35 mV, +40 mV, +45 mV, +50mV, +55 mV, +60 mV, +65 mV, +70 mV,+80 mV, +85 mV, and/or +90 mV. At each voltage, the electrical currentmay be measured and compared with one or more reference electricalcurrents. In some embodiments, a reference electrical current ismeasured in an open, unblocked pore. In some embodiments, a referenceelectrical current is measured in a modified ClyA pore that is bound toa known protein, for example, a protein whose presence or absence willbe determined in solution.

In certain embodiments, the interaction of a protein analyte with amodified ClyA pore provokes current blocks as compared to open, unboundpores. The amplitude of the current block may be measured as theresidual current percentage (IRES) of the open pore current. In someembodiments, characteristic current blocks are used to identify aspecific protein analyte. For example, current blocks may be short,quick current spikes. Current blocks may be transient, or may last formore than 1 minute. Current blocks may be shallow or may be deep. Ashallow current level may be indicated by an IRES value of about41-100%, indicating that the target protein analyte leaves a residualcurrent that is about 41%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% of the open pore current. Conversely, a deep currentlevel may be indicated by an IRES value of about 0-40%, indicating thatthe target analyte leaves a residual current that is about 0%, 5%, 10%,15%, 20%, 25%, 30%, 35%, or 40% of the open pore current. In someembodiments, a current block comprises more than one current level, forexample, a current block may comprise a shallow current level and a deepcurrent level. In one exemplary modified ClyA pore, a target analyte mayprovoke a first current block with an IRES value of about 70% as well asa second current block with an IRES value of about 15%. In certainembodiments, the presence and/or identity of a protein analyte isdetermined by comparing the shallow and deep current levels from theprotein analyte with shallow and deep current levels from a referenceligand. The inter-conversion between shallow and deep current levels maybe compared, for example, IRES values may be compared, the rate ofconversion between shallow and deep current levels may be compared, therelative durations of the shallow and deep currents levels may becompared, and/or the number of shallow and deep current levels may becompared.

For any target protein analyte, when the voltage across the modifiedClyA pore is varied, the percentage of shallow and/or deep currentlevels may also vary. The distribution of shallow and/or deep currentlevels may differ from one target protein analyte to another targetprotein analyte. Thus, in some embodiments, two or more target analytesare distinguished from one another on the basis of theft current levelmeasurements. Two or more target protein analytes may be distinguishedby the intensity and duration of their current blocks and/or by theirdistributions of current levels. For example, a first protein may show alarge decrease in the percentage of shallow levels as the voltage acrossthe pore is increased, while a second protein may show a more gradualdecrease.

In certain embodiments, the two or more target proteins about 95%, 90%,85%, 80%, 75%, or 70% identical. In certain embodiments, the two or moretarget proteins or their subunits are about 65%, 60%, 55%, 50%, or 45%identical. In some embodiments, the two or more target proteins arespecies-specific forms of the same protein.

2. Translocation of Nucleic Adds

One aspect of the present disclosure is a modified ClyA pore that iscapable of binding, detecting, identifying, translocating nucleic acidsand/or modulating transport of nucleic acids. Nucleic acids whichtranslocate through the modified ClyA pores include but are not limitedto DNA, which may or may not carry posttranslational modifications suchas methylation; RNA such as transfer RNA (tRNA), messenger RNA (mRNA),ribosomal RNA (rRNA), small interfering RNA (siRNA), micro RNA (miRNA),small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), extracellularRNA (exRNA), Piwi-interacting RNA (piRNA), and any non-coding RNA(ncRNA). Nucleic acid analogs such as 2′-O-methyl substituted RNA,locked nucleic acid (LNA), morpholino, and peptide nucleic acid (RNA),dideoxynucleotides, etc.

In certain embodiments, the modified ClyA pores are engineered totranslocate DNA such as ssDNA or dsDNA. While ssDNA in particular maynot enter a modified ClyA pore at physiological salt conditions due toreplusive charges from negatively-charged residues in the lumen, DNAtranslocates through modified ClyA pores as described herein underconditions of high ionic strength. In some embodiments, conditions ofhigh ionic strength are (or have the equivalent to the ionic strengthof) a 2.5 M NaCl solution. In certain embodiments, conditions of highionic strength are higher than the ionic strength of a 2.5 M NaCl, forexample, conditions of high ionic strength may be (or may be equivalentto the ionic strength of) a 2.75 M, 3 M, 3.25 M, 3.5 M, 3.75 M, 4 M,4.25 M, 4.5 M, 4.75 M, or 5 M NaCl solution and/or may be (or may beequivalent to) 2.75 M, 3 M, 3.25 M, 3.5 M, 3.75 M, 4 M, 4.25 M, 4.5 M,4.75 M, or 5 M KCl solution. The negatively charged residues lining theinternal lumen of the ClyA pore may be screened under these conditions.For example, in 2.5 M NaCl, 15 mM Tris-HCl at pH 7.5, addition of ds DNAto a modified ClyA pore as described herein at +100 mV may produce ashort current blockade due to translocation of dsDNA through the pore.As demonstrated herein, dsDNA translocates the modified ClyA pores underthese conditions. In addition, ssDNA may translocate the modified ClyApores, for example under conditions of high ionic strength and/or in afolded structure. ssDNA may translocate modified ClyA pores at adifferent rate than dsDNA.

Accordingly, one aspect of the present disclosure is a method fortranslocating DNA, for example dsDNA through a modified ClyA pore thatis capable of translocating DNA, comprising the steps of obtaining amodified ClyA pore as described herein, applying a voltage of at least+50 mV across the modified ClyA pore, adding a sample containing the DNAto the cis opening of the modified ClyA pore, and measuring the currentflowing through the pore. A current blockade indicates translocation ofthe DNA. Current may be restored by reversing the potential to anegative potential, such as −100 mV or −50 mV. In some embodiments, themodified ClyA pore is used under conditions of high ionic strength.

In some embodiments, the modified ClyA pore for translocating DNAcomprises a modified ClyA pore as described herein. The modified ClyApores may be engineered to translocate nucleic acids. For example, themodified ClyA pore may comprise at least 12 subunits, wherein eachsubunit comprises a polypeptide represented by an amino acid sequence atleast 80% identical to SEQ ID NO:1, wherein exactly one Cys residue issubstituted with Ser. Each subunit may be represented by an amino acidsequence that is at least 85%, 90%, 95%, 96%, 96%, 98%, or 99% identicalto SEQ ID NO:1, and additionally exactly one Cys residue may besubstituted with Ser. The Cys residue may be Cys 87 and/or Cys 285 inSEQ ID NO:1. In some embodiments, the Cys residue is Cys285. Other aminoacid residues may be substituted, for example, with amino acids thatshare similar properties such as structure, charge, hydrophobicity, orhydrophilicityo. In certain embodiments, substituted residues are one ormore of L99, E103, F166, and K294. For example, the substituted residuesmay be one or more of L99Q, E103G, F166Y, and K294R. Thus, each subunitmay comprise a polypeptide represented by an amino acid sequence of SEQID NO:2. An exemplary modified ClyA pore comprising subunits in whichexactly one Cys residue is substituted with Ser may be called ClyA-CS.

In some embodiments, the modified ClyA pore recognizes and chaperones aspecific DNA molecule across a biological membrane under a fixedtransmembrane potential. The reaction mechanism may be based on DNAstrand displacement. For example, a DNA moiety may be conjugated to ananopore in order to allow the transport of selected DNA moleculesacross the nanopore via the DNA strand displacement reaction. In certainembodiments, the modified ClyA pore comprises at least 12 subunits,wherein each subunit comprises C875, C285S, and D103C substitutions, andeach subunit is conjugated to an oligonucleotide. Accordingly, in someembodiments, a method for translocating dsDNA comprises (a) obtaining amodified ClyA pore comprising at least 12 subunits, wherein each subunitcomprises C875, C285S, and D103C substitutions and is conjugated to anoligonucleotide; (b) applying a voltage of +50 mV across a modified ClyApore as described herein, (c) adding a sample containing a dsDNA to thecis opening of the modified ClyA pore, (d) adding to the cis opening ofthe modified ClyA pore a first single stranded nucleic acid comprising(i) a sequence that is complementary to at least 15 nucleobases of theoligonucleotide that is conjugated to the ClyA pore, and (ii) a sequenceis that is complementary to at least 12 nucleobases of the doublestranded DNA; and measuring the current across the modified ClyA pore,wherein a decrease in current after step (d) indicates translocation ofthe double stranded DNA through the modified ClyA pore.

In some embodiments, the method optionally comprises adding to a transopening of the modified ClyA nanopore a second single stranded nucleicacid comprising a sequence that is complementary to the first singlestranded nucleic acid. Here, an increase in current across the modifiedClyA pore after adding the second single stranded nucleic acid to atrans opening of the modified ClyA pore indicates that the doublestranded DNA has translocated completely through the pore.

A further aspect of the present disclosure relates to a device fortranslocating DNA, comprising: a fluid-filled compartment separated by amembrane into a first chamber and a second chamber; electrodes capableof applying potential across the membrane; one or more nanoporesinserted in the membrane; a solution of high ionic strength in onechamber of the membrane, wherein DNA translocates through the nanoporefrom the first chamber to the second chamber. In certain embodiments,the DNA is double stranded. The nanopores may be ClyA pores, forexample, the modified ClyA pores described herein. The pores, such asmodified ClyA pores, may have an inner diameter of at least 2.2 nm. Insome embodiments, the membrane is an artificial lipid bilayer. In someembodiments, the potential across the membrane ranges from −100 mV to+100 mV. The solution of high ionic strength may comprise 2.5M NaCl.

EXAMPLES

Having provided a general disclosure, the following examples help toillustrate the general disclosure. These specific examples are includedmerely to illustrate certain aspects and embodiments of the disclosure,and they are not intended to be limiting in any respect. Certain generalprinciples described in the examples, however, may be generallyapplicable to other aspects or embodiments of the disclosure.

Example 1 Tuning the Property of ClyA by Directed Evolution

Directed evolution approaches for tailoring enzymes with desiredproperties were used to improve the activity of ClyA-SS, reasoning thatmutations that compensate for the deleterious effects of C875 and C285Ssubstitutions would also increase stability of the nanopore in lipidbilayers. Random libraries were generated on the background of ClyA-SSby error prone PCR (approximately 1-4 mutations per gene per round) andscreened for hemolytic activity (FIG. 6). The most active variants werethen purified by Ni-NTA affinity chromatography and tested for oligomerformation by BN-PAGE (FIG. 7). Selected nanopore variants were finallyscreened in planar lipid bilayers for the desired behavior (lowelectrical noise and ability to remain open at high applied potentials),which served as final and critical criteria for selection. After justfour rounds of screening, ClyA-CS variants were isolated (Table 1) thatshowed low electrical noise (FIG. 8) and remained open in planar lipidbilayers from +90 to −150. Remarkably, the serine at position 87converted back to cysteine, the original residue in the wild-type gene.In order to obtain a cysteine-less ClyA variant amenable tosite-specific chemical modification, ClyA-CS was subjected to saturationmutagenesis at position 87, the resulting library was screened, andcysteine-less ClyA-AS with desired electrical properties (SI) wasselected. In contrast to ClyA-SS, evolved ClyA nanopores expressed in E.coli cells in the soluble fraction (FIG. 6) and the monomers could bepurified in one-step by affinity chromatography, which allowed a tenfold increase in the production yield (˜0.6 mg per 10 ml culture).

Example 2 Isolation of ClyA Nanopores with Different Size

ClyA oligomers, formed by incubation of ClyA monomers with 0.5% w/vβ-dodecyl maltoside (DDM), revealed multiple bands on a BN-PAGE (FIG. 2a), suggesting that ClyA might assemble into several oligomeric states.This is particularly intriguing, since the exact stoichiometry of E.coli ClyA oligomerisation is controversial. The ClyA crystal structure(PDB_ID: 2WCD) revealed a dodecamer with a 5.5 nm opening on the cisside and a 3.3 nm opening at the trans entrance (including Van der Waalsradii of the amino acid side chains), while earlier cryo-EM structuresrevealed nanopores with 8¹¹ or 13¹² subunits.

In planar lipid bilayers, ClyA-WT pre-incubated with DDM showed a widedistribution of open nanopore conductances spanning approximately 2 nS(FIG. 2 b, top), suggesting that also in lipid bilayers ClyA-WT mightassemble into nanopores of different size and/or geometry. The majorpeak in the distribution of ClyA-WT unitary conductance (Type I ClyA)represented only 24% of the reconstituted nanopores and showed anaverage conductance of 1.83±0.06 nS in the conductance range from 1.7 to1.9 nS (−35 mV, 15 mM Tris.HCl pH 7.5 and 150 mM NaCl). The distributionof ClyA-CS open pore conductance showed two major peaks: the firstincluded 37% of the reconstituted nanopores and corresponded to Type IClyA-CS (1.79±0.05 nS); while the second (Type II ClyA-CS) included 23%of the nanopores and showed an average conductance of 2.19±0.09 nS(conductance range 2.1-2.4 nS, FIG. 2 b, middle). ClyA-WT and ClyA-ASalso showed small percentage of Type II ClyA (18% and 16%,respectively). The unitary conductance of ClyA-AS was especially uniformwith 52% of the reconstituted nanopores corresponding to Type I ClyA(FIG. 2 b, bottom).

To establish whether the different bands of ClyA oligomers correspondedto nanopores with different size, ClyA-CS were extracted from the threemajor oligomeric bands in the BN-PAGE, and measurements were made of theunitary open nanopore conductance of 62 nanopores derived from each bandwithin two days from gel extraction. 62% of ClyA-CS oligomers from thelowest band formed Type I ClyA-CS nanopores (1.78±0.04 nS, FIG. 3a ),while 68% of nanopores extracted from the second lowest band (FIG. 3b )reconstituted as Type II ClyA-CS nanopores (2.19±0.09 nS).Interestingly, 42% of the nanopores extracted from the third bandreconstituted in lipid bilayers as a third nanopore type (Type III ClyA)that showed an average conductance of 2.81±0.11 nS in the conductancerange 2.5-3.0 nS (FIG. 3c ).

Taken together, these results show that the three major bands of ClyAoligomers observed on the BN-PAGE correspond to three distinct nanoporetypes with different size and different unitary conductance. Thisfinding is consistent with reports that high order symmetricaloligomeric structures are often permissive with respect to subunitstoichiometry.¹³ Therefore it can be hypothesized that Type I ClyA mostlikely represents the 12 mer of the crystal structure, while Type IIClyA might be the 13 mer observed in earlier cryo-EM studies. Bothnanopores showed low electrical noise (FIG. 8) and remained open over awide range of applied potentials (from +90 mV to −150 mV). Type IIIClyA-CS nanopores, which showed higher noise than Type I and Type IInanopores (FIG. 8) and frequently gated especially at applied potentialslower than −40 mV and higher than +50 mV, may correspond to a 14 merversion of ClyA not observed before.

Example 3 HT as Molecular Caliper to Test ClyA Nanopores of DifferentSize

The ability to employ nanopores with identical amino acid compositionbut different size is a new feature in the biological nanopore field andis important because the size of a nanopore defines its ability tocapture and study a particular molecule.^(10b, 14) It has beenpreviously shown^(7a) that at −35 mV HT (human thrombin, 37 kDa)inflicted well-defined current blockades to Type I ClyA-SS nanoporesthat lasted for several minutes. The blockade signal switched rapidlybetween two current levels, level 1 [percentage of the open nanoporecurrent (I_(RES%))=56±1%] and level 2, (I_(RES%)=23±1%, Table 1, andFIG. 4a ), reflecting two residence sites for HT within the lumen of theClyA nanopore. Level 2 is most likely associated to HT residence at adeep site, while level 1 is associated to the residence of HT closer tothe cis entrance of the nanopore.^(7a) Because thrombin provoked such awell-defined pattern of current blockades HT was used here as amolecular caliper to compare the geometries of the different ClyAnanopores.

At −35 mV HT current blockades to Type I ClyA-CS nanopores wereidentical to that of Type I ClyA-SS nanopore (Table 1), confirming thatmutations accumulated in the variants disclosed herein most likely didnot change the size and geometry of the ClyA nanopore. HT currentblockades to Type II ClyA also switched between the two current levels,but their relative distribution was different. In Type I ClyA-CS HTmostly lodged at the more superficial binding site (70% occupancy),while in Type II ClyA-CS HT preferred the binding site deeper within thenanopore (96% occupancy). These results suggest that both ClyA Typesmost likely retain similar overall nanopore architecture but providedifferent steric hindrance to HT. HT blockades to Type III ClyA werefast (55±48 ms) and showed only a level 2 current block (I_(RES%) of32±9%), suggesting that Type III ClyA is large enough to allowunhindered translocation of HT through the nanopore (see below).

Example 4 Protein Translocation Through ClyA Nanopores

Thrombin is a globular protein with a molecular volume that can beapproximated to a sphere with diameter of 4.2 nm (SI). Therefore,assuming that Type I, II and III ClyA correspond to nanopores withdifferent oligomeric state (see above), HT should not easily translocatethrough Type I and Type II ClyA nanopores (trans diameter, including theVan der Waals radii of the atoms, of 3.3 and 3.7 nm, respectively, Table1). Contrary, HT has the same diameter as Type III ClyA (Table 1),suggesting that the protein might be capable of translocating throughthis nanopore. A powerful method to assess whether molecules passthrough nanopores is to investigate the voltage dependence of theduration of the proteins current blockades. The decrease of the durationof the current blockades with increasing potential is strong evidencethat the molecules translocate through the nanopore. By contrast, anincrease in the duration of the current blockades with the voltagesuggests that the proteins are driven into the nanopore but do nottranslocate through it.^(10f, 15) From −5 mV to −25 mV the dwell time ofHT blockades to Type I ClyA-SS nanopores increased with the appliedpotential (FIG. 9a ), suggesting that HT does not translocate throughthe nanopores in this voltage interval. Taking advantage of the factthat Type I and Type II evolved ClyA nanopores remained open at appliedpotentials up to −150 mV, HT blockades were characterized on Type I andII ClyA-CS nanopores from −60 to −150 mV. At applied potentials higherthan −100 mV for Type I ClyA-CS nanopores and −60 mV for Type II ClyA-CSnanopores the dwell times of HT current blockades strongly decreasedwith increasing potential (FIG. 5a ), suggesting that in this potentialrange HT molecules translocate through the nanopores. Similarly,Dendra2_M159A (FP, a GFP like protein, 30 kDa protein) displayed aninitial increase (from −25 mV to −40 mV) followed by a decrease of theduration of the current blockades (from −50 mV to −70 mV), suggestingthat FP translocates through Type I ClyA-SS nanopores at potentialshigher than −50 mV (FIG. 9b ). Comparing the duration of HT blockades toType I and Type II ClyA-CS nanopores at the same potential revealed thatthe translocation of HT through Type II ClyA-CS was about two orders ofmagnitude faster than for Type I ClyA-CS (FIG. 5 b, Table 1), which isin line with the interpretation that Type I and Type II ClyA describenanopores with different size.

Example 5 Folded Versus Unfolded Translocation

When a molecule is lodged within the lumen of a nanopore, the ioniccurrent block is proportional to the atomic volume of the electrolytesbeing excluded by the molecule.^(10d, 10f, 16) Therefore, if themolecule translocates through the nanopore with a folded structure, theI_(RES%) should remain constant at different applied voltages. Incontrast, if a protein unfolds upon translocation, the I_(RES%) isexpected to change, giving that the volume and shape of the unfoldedpolypeptide chain is different to that of the globular protein. TheI_(RES%) values during the translocation of HT through Type I and TypeII nanopores were identical at −35 mV and −150 mV (level 2, Table 1),suggesting that in this potential range HT does not unfold while in thenanopore. Interestingly, solely with Type I ClyA-CS nanopores, thecurrent blockades of HT at potentials below −90 mV often terminated witha current block of higher I_(RES%) (shoulder) followed by a currentincrease (spike) with respect to the open nanopore current (FIGS. 5c and10). Although the shoulder in the I_(RES%) values might indicate that HTunfolds upon translocation, the current spikes that follows the proteintranslocation suggest otherwise that ClyA nanopores may need to deformin order to allow the translocation of folded HT through Type Inanopores.

Example 6 Translocation of dsDNA Through a ClyA Pore

In this work ClyA-CS was selected for its enhanced activity, solubilityand favourable behaviour in planar lipid bilayers when compared to WildType ClyA The internal diameter of the ClyA dodecamers (3.8 nm at itsnarrower entrance,¹⁷ FIG. 11a ) is larger than the diameter of dsDNA(2.2 nm for the B form), indicating that dsDNA should readilyelectrophoretically translocate through the pore. However, most likelybecause of the negatively charged residues lining the lumen of ClyA(pl=5.1), at physiological salt concentrations ssDNA does not enter thenanopore.^(7a) In view of previous work with alpha hemolysin (αHL)nanopores at high alkaline PH,^(7b, 7c) the ability of DNA totranslocate through ClyA-CS nanopores was tested at high ionic strength,where the internal charges of the pore are screened. In 2.5 M NaCl, 15mM Tris.HCl pH 7.5 and under +100 mV applied potential, the addition of0.12 μM of biotinylated dsDNA 1 (290 bp, Table 2) to the cis compartmentproduced transient current blockades (I₈) to the open pore current(I_(O)) showing a residual current (I_(RES)=I_(B)/I_(O)) of 0.63±0.01(level 1*₊₁₀₀=1.10±0.03 nA, n=3 experiments), with 2.0±0.6 ms dwelltime, due to the entrance of the DNA into the lumen of the pore (FIG.11b ). The subsequent addition to the cis compartment of 0.3 μM ofneutravidin, which forms a tight complex with biotin, converted thetransient blockades into long lasting current blockades (level1₊₁₀₀=1.19±0.01 nA, n=4) with a higher residual current value(I_(RES)=0.68±0.01). The open pore current could be restored by reversalof the applied potential to −100 mV (FIG. 11b ). These results suggestthat neutravidin prevents the full translocation of DNA through ClyAnanopores by forming a cis protein:DNA complex where the DNA occupiesthe full length of the pore (FIG. 11c ). Trans complexes could also beformed at −100 mV (level 1₊₁₀₀=1.02±0.03 nA, I_(RES)=0.62±0.01, n=4)when the dsDNA:neutravidin complex is threaded through the trans side(FIG. 11d ).

Example 7 A Rotaxane System Traps a dsDNA Within a ClyA Nanopore

Rotaxanes are supramolecular interlocked systems in which a linear unit(thread) is translocated through a microcyclic ring and is tapped by twobulky substituents (stoppers). Such mechanically joined molecules haveapplications for example as switches in molecular electronics or ascomponents in molecular machineries. Rotaxanes have been made from avariety of molecules including dsDNA²² or by locking a biotinylatedssDNA molecules threaded through αHL nanopore with streptavidin on oneside and with a DNA hairpin on the other side (dsDNA can not translocatethrough αHL).²³ Here to prove the translocation of dsDNA through ClyAnanopores a rotaxane system was built in which a dsDNA molecule added tothe trans side of a ClyA nanopore hybridize with a second DNA strand onthe cis side after threading through the lumen of the nanopore. A ClyAnanopore ClyA-2 was used, which contains 12 ssDNA molecules 2 (51 bases,Table 2, FIG. 12a ) covalently attached at their 5′ ends to cysteineresidues introduced at the cis entrance of the pore (at position 103,FIG. 11a ) via disulphide linkages. 2 is designed to act as a rotaxanestopper. The thread 3 is a dsDNA molecule (59 bp, Table 2) with anadditional 31 nucleobases stretch of ssDNA at the 5′ end that isdesigned to hybrize with the stopper at the cis side throughhybridisation with oligo 6; and a 3′ biotinylated linker that is usedfor complexation with neutravidin at the trans side. The linkage betweenthe thread and stopper on the cis side is mediated by a bridging ssDNAmolecule 4 (Table 2, FIG. 12a ) that is complementary to the first 16nucleobases of 2 and to the last 25 nucleobases of 3. When 3 and 4 areadded to the trans compartment, at −100 mV the DNA thread is captured bythe pore and not released from the pore upon reversing of the potentialto +100 mV (level 2₊₁₀₀, I_(RES)=0.77±0.04, n=4), indicating that a DNArotaxane is formed (FIG. 12b ). Interestingly, at +100 mV the residualcurrent of the rotaxane was higher than the I_(RES) values of the cis ortrans pseudorotaxane threads (0.68±0.01 and 0.62±0.01, respecively),suggesting that an unhybridized ssDNA stretch of 2 is likely to span thepore at this potential (FIG. 12b ). The rotaxane could be disassembledby addition of 20 mM DTT to the cis chamber, which reduced thedisulphide bond between 2 and ClyA and restored the open pore current at+100 mV (FIG. 12c-12e ).

Selective Translocation Through ClyA-2 Pores.

The data indicate that ClyA-2 excludes non-specific DNA from the porelumen of the pore, suggesting that the mesh ssDNA molecules attached tothe pore might produce a steric and or electrostatic impediment fornon-tethered DNA translocation. In addition, the DNA attached to thepore often occupies the pore lumen (FIG. 15c ) thus preventing theentrance of other DNA molecules. When a specific DNA molecule ishybrised to ClyA-2, rapid DNA capture is observed at positive appliedpotentials. The concentration of ssDNA attached to the pore waspreviously estimated to be ˜20 mM.²¹ Therefore, augmented concentrationof the dsDNA in the proximity of the pore mouth may enhance the captureof the specific DNA strands. In this case, unhybridized strands 2 mightstill be tethered to the pore, in which case the dsDNA construct mighthave to compete with the ssDNA 2 to enter the pore lumen.

Example 8 A Nanopore:DNA Device Utilizing a Strand Displacement Reaction

At high positive applied potentials, the ionic current of ClyA-2nanopores fluctuated between the open pore level and several blockedpore levels (FIG. 12c , FIGS. 15 and 16 a), suggesting the ssDNAmolecules tethered to the top of the pore enter the lumen of ClyA but donot permanently thread to the trans side of the pore.^(1c) Furthersuggesting this interpretation; at +100 mV the addition of a 90 merssDNA 5a (Table 2) in complex with neutravidin to the cis side ofClyA-CS provoked transient current blockades (FIG. 13) that convertedinto long lasting DNA translocation events upon the subsequent additionof equimolar concentrations of the complimentary ssDNA 5b (Table 2, FIG.13). The translocation of DNA through nanopores is often observed abovea threshold potential²⁴⁻²⁷ that can be tuned by modulating the chargedistribution of the lumen of the pore,^(26,27) or by changing the ionicstrength of the solution.^(7c, 28) Therefore, most likely because of itslower charge density and/or higher flexibility, these findings indicatethat ssDNA has higher threshold for DNA translocation than dsDNA (FIG.13).

These results suggest that despite the applied potentials a ssDNAmolecule attached to the cis entrance of ClyA is likely to sample thecis solution. On the other hand if the DNA molecule becomes doublestranded (e.g. by strand hybridisation) at positive applied potentialthe dsDNA strand will translocate through the pore and sample the transsolution. Therefore, a nanopore:DNA device was designed in which thehybridisation of a specific DNA strand to the cis side of the nanoporepromotes the translocation of the DNA hybrid through the pore. TheDNA:nanopore complex is then disassembled by a strand displacementreaction (FIG. 14a ), which will promote the transport of DNA across thebilayer and the return of 2 to sample the cis chamber. Conveniently, atpositive applied potentials the addition of dsDNA molecules to the cisside of a ClyA-2 do not produce current blockades (FIG. 18), indicatingthat the ssDNA molecules attached to ClyA-2 prevent or drasticallyreduce the translocation of DNA from solution. Therefore, the DNA unitatop of the nanopore might infer specificity to the system by promotingthe translocation of specific DNA molecules while creating a barrier forthe translocation of non-specific DNA. At +50 mV, the addition of 3 tothe cis side of ClyA-2 did not produce current blockades, furtherconfirming that the ssDNA molecules attached to ClyA-2 prevent the DNAin solution from entering the pore (FIG. 14b ). Nonetheless, after theaddition to the cis chamber of 6, which is complementary to the first 15bases of 2 and to the last 12 nucleobases of 3 (Table 2), the dsDNAhybrid nanopore showed permanent current blockades withI_(RES)=0.70±0.02 (level 2₊₅₀=0.59±0.02 nA, FIG. 14 c, n=5), thehallmark of DNA capture. The translocation of 3 to the trans side wasconfirmed by the formation of a rotaxane upon addition of neutravidin tothe trans chamber (FIG. 14c -d, FIG. 17e-f ). Crucially, 6 was designedto include a 10 nucleobases 5′-single-strand extension to serve astoehold for the dissociation of the rotaxane (FIG. 14a ). Notably, theaddition to the trans chamber of 7, a ssDNA molecule complementary to 6(Table 2), released 3 from the nanopore by first hybridising to thetoehold and then promoting strand displacement (FIG. 14a ). This wasobserved by the restoration of the open pore current, because the ssDNAmolecule tethered to ClyA returned to the cis side after 3 and 6 arereleased from the pore (FIG. 14d ). Remarkably, the nanopore showed asuccession of open and blocked current levels, reflecting a cycle ofcapture, translocation and release, as the DNA cargos are captured fromthe cis chamber, transported through the pore and released to the transchamber (FIG. 14d ).

The DNA strands at the top of ClyA-2 nanopores drastically reduced thecapture of non-specific DNA at both +50 and +100 mV (FIG. 18),suggesting that the DNA at the top of the pore prevented thetranslocation of dsDNA from solution. Notably, the applied potential wasset to +50 mV and not at +100 mV because at +100 mV ClyA-2 occasionallyproduced long current blockades that were similar to typical eventsprovoked by the capture of non-specific DNA (FIG. 18). Such currentblockades were less frequent at lower applied potentials; hence theexperiment was performed at +50 mV (FIG. 14) and +35 mV (FIG. 20). Anadditional reason to work at lower applied potential, as explained inthe legend of FIG. 14, is that the frequency of DNA capture is reducedwith the potential, thus at lower potentials the cycles of capture andrelease are more easily observed.

ssDNA vs dsDNA Translocation

In the rotaxane configuration ssDNA molecules may be spanning the entirelength of the pore at positive applied potentials. This is likelybecause at +100 mV the I_(RES) value of the rotaxane (0.77) is higherthan the I_(RES) values of the cis- and trans-pseudorotaxanes (0.68 and0.62, respectively, FIGS. 11c and 11d ), which have a dsDNA immobilisedwithin the pore. In addition, the unitary conductance values of therotaxane as calculated from the slopes of the I-V curves were lower atpositive applied potentials (10.8 nS) than at positive bias (13.0 nS,FIG. 16). Since ssDNA has a diameter (d=1 nm) is smaller than dsDNA(d=2.2 for the B form), these results further suggesting that ssDNAoccupies the pore at positive bias. To further investigate the abilityof ssDNA to span the pore, the ability of DNA hybrid 3, which is formedby a 3′ biotinylated dsDNA section of 59 nucleobasepairs followed by assDNA stretch of 31 nucleobases at the 5′ end, was tested for ability totranslocate through ClyA-CS pores. At +100 mV, the addition of 3 incomplex with neutravidin to the cis side of a ClyA-CS pore provoked longlasting current blockades with I_(RES)=0.67 (FIG. 19), the same I_(RES)of a cis-pseudorotaxane (0.68), suggesting that at this potential theDNA hybrid translocate through ClyA and dsDNA spans the lumen of thepore. Since the translocation of 3 can only be initiated from the ssDNAend these results suggest that at +100 mV the ssDNA leading sequence iscapable of translocating the lumen of ClyA. Interestingly, at +50 mV and+70 mV the current blockades were only transient (FIG. 19), suggestingthat at this voltage ssDNA cannot pass the ClyA pore. The addition ofstrand 6, which is complimentary to the last 12 nucleobases of 3produced long lasting current blockades +70 mV (FIG. 19), suggestingthat threshold for DNA translocation is reduced.

Materials & Methods Screening of ClyA Nanopores.

ClyA was expressed in E. cloni® EXPRESS BL21 (DE3) cells (Lucigen) byusing a pT7 plasmid. Transformants were prescreened on Brucella Agarwith 5% Horse Blood (BBL™, Becton, Dickinson and Company), andindividually grown and overexpressed in 96-deep-wells plates. Monomersfrom cell lysates were first screened for hemolytic activity on horseerythrocytes (bioMérieux) and then purified by using Ni-NTA affinitychromatography. Purified monomers were oligomerized in the presence of0.5% β-dodecyl maltoside (GLYCON Biochemicals GmbH)¹² and loaded onnative gel electrophoresis gels to check for oligomerisation. Theelectrical properties of ClyA oligomers were then screened in planarlipid bilayers.

Purification of Evolved ClyA Nanopores.

ClyA was expressed in E. cloni® EXPRESS BL21 (DE3) cells by using a pT7plasmid. Monomers were purified by using Ni-NTA affinity chromatographyand oligomerized in the presence of 0.2% β-dodecyl maltoside (GLYCONBiochemicals GmbH).

Electrical Recordings.

Ionic currents were measured by recording from planar bilayers formedfrom diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids,Alabaster, Ala.). Currents were measured with Ag/AgCl electrodes byusing a patch-clamp amplifier (Axopatch 200B, Axon Instruments, FosterCity, Calif.).¹⁸

Construction of ClyA Libraries by Error-Prone PCR.

Libraries were constructed by amplifying the ClyA genes from plasmid DNAusing T7 promoter and T7 terminator primers (Table 3). In the firstmutagenesis round we used as a template a plasmid containing thesynthetic gene encoding for ClyA-SS from Salmonella Typhi. From thesecond mutagenesis round we used the DNA plasmids that were derived fromthe previous round of selection. In ClyA-SS, the WT sequence wasmodified by the substitution of the two Cys residues (positions 87 and285) with Ser and by the attachment of DNA encoding a Gly-Ser-Ser linkerfollowed by a C-terminal hexahistidine tag.^(7a)

DNA amplification was performed by error prone PCR: 400 μL of the PCRmix (200 μl of REDTaq ReadyMix, 8 μM final concentration of forward andreverse primers, ˜400 ng of plasmid template and ddH₂O up to 400 μl) wassplit into 8 reaction mixtures containing 0-0.2 mM of MnCl₂ and cycledfor 27 times (pre-incubation at 95° C. for 3 min, then cycling:denaturation at 95° C. for 15 sec, annealing at 55° C. for 15 sec,extension at 72° C. for 3 min). These conditions typically yielded 1-4mutations per gene in the final library. The PCR products were pooledtogether, gel purified (QIAquick Gel Extraction Kit, Qiagen) and clonedinto a pT7 expression plasmid (pT7-SC1) by MEGAWHOP procedure:¹⁹ ˜500 ngof the purified PCR product was mixed with ˜300 ng of ClyA-SS circularDNA template and the amplification was carried out with Phire Hot StartII DNA polymerase (Finnzymes) in 50 μL final volume (pre-incubation at98° C. for 30 s, then cycling: denaturation at 98° C. for 5 sec,extension at 72° C. for 1.5 min for 30 cycles). The circular templatewas eliminated by incubation with Dpn I (1 FDU) for 2 hr at 37° C. Theresulted mixture was desalted by dialysis against agarose gel (2.5%agarose in Milli-Q water) and transformed into E. cloni® 10G cells(Lucigen) by electroporation. The transformed bacteria were grownovernight at 37° C. on ampicillin (100 μg/ml) LB agar pates typicallyresulting in >10⁵ colonies, and were harvested for library plasmid DNApreparation.

Construction of Saturation Mutagenesis Library at Position 87 of EvolvedClyA

In order to have a library containing cysteine-free ClyA variants, thegene encoding for 4ClyA4 (Table 2) was amplified using the 87NNS primer(containing a degenerate codon at position 87 encoding for the completeset of amino acids, Table 3) and T7 terminator primers. PCR conditions:0.3 mL final volume of PCR mix (ReadyMix®), containing ˜400 ng oftemplate plasmid, cycled for 30 times (pre-incubation at 95° C. for 3min, then cycling: denaturation at 95° C. for 15 sec, annealing at 55°C. for 15 sec, extension at 72° C. for 3 min). The resulting PCR productwas cloned into pT7 expression plasmid by MEGAWHOP procedure using4ClyA4 circular template (see above).

Construction of the ClyA-WT

ClyA-SS gene was amplified using 87C and 285C primers (Table 3). PCRconditions: 0.3 mL final volume of PCR mix (150 μl of REDTaq ReadyMix, 6μM of forward and reverse primers, ˜400 ng of template plasmid), cycledfor 27 times (pre-incubation at 95° C. for 3 min, then cycling:denaturation at 95° C. for 15 sec, annealing at 55° C. for 15 sec,extension at 72° C. for 3 min). The resulting PCR product was clonedinto pT7 expression plasmid by the MEGAWHOP procedure described above,using ClyA-SS circular template.

Screening of ClyA Libraries and Hemolytic Assay

Since ClyA-SS displays “border of detection” hemolytic activity, duringthe first two rounds of the mutagenesis libraries were only screened foractivity on Brucella Agar with 5% Horse Blood. From the third selectionround, colonies displaying hemolytic activity on Brucella Agar werefurther screened for hemolytic activity on horse erythrocytes from thecrude lysate after overexpression. The goal of this work was to obtainClyA variants that oligomerise well in β-dodyl maltoside (DDM) and formnanopores with low electrical noise and uniform unitary conductance inlipid bilayers. Therefore, screening for hemolytic activity alone couldnot serve as the sole criteria for selection of such variants (forexample WT-ClyA is very hemolytically active, but shows non-uniformunitary current distribution). Thus, from the 4th round, proteins fromthe crude lysate were purified by Ni-NTA affinity chromatography and theoligomerisation of ClyA was tested on BN-PAGE after incubation in DDM.ClyA nanopores that oligomerised well were then tested in planar lipidbilayers (with particular stress on uniformity of formed channels, lowelectrical noise and stability at high applied potentials).

Screening for Hemolytic Activity on Brucella Horse Blood Agar Plates

After the plasmid DNA was electroporated into E. cloni® EXPRESS BL21(DE3) cells (Lucigen), transformants were prescreened on Brucella Agarwith 5% Horse Blood (BBL™, Becton, Dickinson and Company) supplementedwith 100 μg/ml ampicillin. Clones displaying hemolytic; activity, whichwas observed by a lytic aura around the colonies after overnight growthat 37° C., were individually grown in 96-deep-wells plates overnight byshaking at 37° C. (0.5 ml 2xYT medium containing 100 μg/mL ampicillin).The obtained cultures were either pooled for preparation of plasmid DNA(QIAprep, Qiagen) that served as a template for the next round (rounds 1and 2), or used as starters for protein overexpression (rounds 3 to 5).

Screening for Hemolytic Activity of Crude Lysates After ClyAOverexpression

Rounds 3 to 5: 50 μl of the starter cultures from 400-600 clones (seeabove) were inoculated into 450 μl of fresh medium in new 96-deep-wellsplates and the cultures were grown at 37° C. until OD₆₀₀˜0.8. Then IPTG(0.5 mM) was added to induce overexpression, and the temperature wasreduced to 25° C. for an overnight incubation. Next day, bacteria wereharvested by centrifugation at 3000× g for 15 min at 4° C., thesupernatant was discarded and pellets were incubated at −70° C. for fewa hours to facilitate cell disruption. Then pellets were resuspended in0.3 mL of lysis buffer (15 mM Tris.HCl pH 7.5, 150 mM NaCl, 1 mM MgCl₂,10 μg/ml lysozyme and 0.2 units/mL DNAse I) and lysed by shaking at 1300RPM for 30 min at 37° C. 5-30 μL of lysates were then added to 100 μL of˜1% horse erythrocytes suspension. The latter was prepared bycentrifuging horse blood (bioMérieux) at 6000× g for 5 ruins at 4° C.,the pellet was resuspended in 15 mM Tris.HCl pH 7.5, 150 mM NaCl (If thesupernatant showed a red color, the solution was centrifuged again andthe pellet resuspended in the same buffer). The hemolytic activity wasmonitored by the decrease in OD at 650 nm over time (˜3-10 minintervals, measured using Multiskan GO Microplate Spectrophotometer,Thermo Scientific). The hemolytic activity of the clones was comparedwith the activity of 2-4 parent clones from the previous round that weregrown in the same plates as a reference.

Screening for Oligomerization and Nanopore Formation of EvolvedVariants.

Rounds 4: 6-12 of the most hemolytically active variants were partiallypurified from the same lysates that were used for the screening ofhemolytic activity by using Ni-NTA affinity chromatography: 0.2 mL ofcrude lysate containing monomeric ClyA was brought to 1 mL with 15 mMTris.HCl pH 7.5, 150 mM NaCl supplemented with 1% DDM (to trigger ClyAoligomerization) and 10 mM imidazole, incubated at ambient temperaturefor 20 min and centrifuged at 20′000× g for 10 min at 4° C. Theclarified lysates were avowed to bind to 20 μL (bead volume) of Ni-NTAagarose beads (Qiagen) for 1 hr by gentle mixing at 4° C. The unboundfraction were removed by centrifugation at 20′000× g for 10 min at 4° C.(the supernatant was discarded). Finally, oligomerized ClyA proteinswere eluted with 50 μL of 600 mM imidazole in 15 mM Tris.HCl pH 7.5 150mM NaCl 0.2% DDM. Typically 40 μg of ClyA were supplemented with ˜10%glycerol and 1× of NativePAGE™ Running Buffer and 1× Cathode BufferAdditive (Invitrogen™) and then loaded on BN-PAGE (FIG. 7). Variantsthat formed oligomers on BN-PAGE were then tested in planar lipidbilayers (with particular stress on uniformity of formed channels).

In round 5 the ClyA-CS clones that were subjected to saturationmutagenesis at position 87 were screened in order to obtain acysteine-less ClyA variant amenable to site-specific chemicalmodification. Therefore, since that clones containing cysteine atposition 87 were expected to show the highest activity, in this roundthe clones that showed medium to high hemolytic activity in the crudelysates were isolated. ClyA variants were then partially purified andselected as explained above.

Proteins Overexpression and Purification

E. cloni® EXPRESS BL21 (DE3) cells were transformed with the pT7 plasmidcontaining the ClyA gene. Transformants were grown overnight at 37° C.on LB agar plates supplemented with 100 mg/L ampicillin. The resultingcolonies were pooled together and innoculated into 50 mL of LB mediumcontaining 100 mg/L of ampicillin. The culture was grown at 37° C., withshaking at 200 rpm, until it reached an OD₆₀₀ of 0.6 to 0.8. Theexpression of ClyA was then induced by the addition of 0.5 mM IPTG andthe growth was continued at 20° C. The next day the bacteria wereharvested by centrifugation at 6000× g for 10 min and the pellets werestored at −70° C.

The pellets containing monomeric ClyA were thawed and resuspended in 20mL of wash buffer (10 mM imidazole 150 mM NaCl, 15 mM Tris.HCl, pH 7.5),supplemented with 1 mM MgCl₂ and 0.05 units/mL of DNAse I and thebacteria were lysed by sonication. The crude lysates were clarified bycentrifugation at 6000× g for 20 min and the supernatant was mixed with200 μL of Ni-NTA resin (Qiagen) pre-equilibrated with wash buffer. After1 h, the resin was loaded into a column (Micro Bio Spin, Bio-Rad) andwashed with ˜5 ml of the wash buffer. ClyA was eluted with approximately˜0.5 mL of wash buffer containing 300 mM imidazole. Proteinconcentration was determined by Bradford assay and the purified proteinswere stored at 4° C.

Construct of ClyA models

The 13 mer and 14 mer ClyA nanopores were modeled from the 12 mer of thecrystal structure (PDB code: 2WCD) as follow: A central axis wasconstructed through the length of the 12 mer (ax12), and the distancebetween the C-alpha atom of residue 114 in the monomer A (114Ca-A) andthe ax12 was measured giving the approximate radius of the pore (r12).The distance (d) between the 114Ca-A and the equivalent atom in monomerB (114Ca-B) was used to calculate the approximate circumference of the12 mer (c12=d×13), 13 mer (c13=d×13) and 14 mer (c14=d ×14). The radiusof the three oligomers (r12, r13 and r14) was then calculated from thecircumference using simple trigonometry. The 12 mer, 13 mer and 14 merwere then built by placing the monomers at distances r12, r13 and r14,respectively, from the central ax and rotated over an angle of 360°/12,360°/13 and 360°/14, respectively. The 12 mer that was bunt using thismethod reproduced perfectly the 12 mer of the X-ray crystal structure(RMS=0.29 Å), showing the high degree of symmetry and feasibility toconstruct higher order pores.²⁰

The size of the nanopore opening was obtained by increasing the Van derWaals radii of the atoms of ClyA until the pore closed. Then theincreased value of the Van der Waals radii was taken as the radius ofthe pore. The diameter of thrombin was calculated from a spherecorresponding to the measured molecular volume of the protein.²⁰

Electrical Recordings in Planar Lipid Bilayers.

The applied potential refers to the potential of the trans electrode.ClyA nanopores were inserted into lipid bilayers from the ciscompartment, which was connected to the ground. The two compartmentswere separated by a 25-μm thick polytetrafluoroethylene film (GoodfellowCambridge Limited) containing an orifice ˜100 μm in diameter. Theaperture was pretreated with ˜5 μL of 10% hexadecane in pentane and abilayer was formed by the addition of ˜10 μL of1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in pentane (10mg/mL) to both electrophysiology chambers. Typically, the addition of0.01-0.1 ng of oligomeric ClyA to the cis compartment (0.5 mL) wassufficient to obtain a single channel. Electrical recordings werecarried out in 150 mM NaCl, 15 mM Tris.HCl pH 7.5. The temperature ofthe recording chamber was maintained at 28° C. by water circulatingthrough a metal case in direct contact with the bottom and sides of thechamber.

Data Recording and Analysis

Electrical signals from planar bilayer recordings were amplified byusing an Axopatch 200B patch damp amplifier (Axon Instruments) anddigitized with a Digidata 1440 A/D converter (Axon Instruments). Datawere recorded by using Clampex 10.2 software (Molecular Devices) and thesubsequent analysis was carried out with Clampfit software (MolecularDevices). Open pore currents and HT blockades were recorded by applyinga 10 kHz low-pass Bessel filter and sampling at 50 kHz if not otherwisestated. For unitary channel conductance distributions data collection,traces were further filtered digitally with Gaussian low-pass filterwith 200 Hz cutoff. The open pore currents were determined for allinserted channels at both +35 and −35 mV to ensure that the poreinserted with the correct orientation (the unitary conductance of ClyAis higher at positive applied potentials when reconstituted from the cisside) and values corresponding to −35 mV were used to construct thedistributions. Open pore current values (I_(O)) for ClyA and blockedpore current values (I_(B)) for HT were calculated from Gaussian fits toall points histograms (0.3 pA bin size).³ Histograms for HT and BTblockades were prepared from at least 10 current blockade at least 0.5 slong. The residual current values (I_(RES)) were calculated as:I_(RES%)=I_(B)/I_(O)%. When HT produced two current levels within thesame blockade, theft relative contributions (FIG. 4 b, Table 1) werededuced from the area of the peaks obtained from Gaussian fits to theall points histogram.³ HT blockade lifetimes were calculated by fittingthe cumulative distribution of the block dwell times for at least 50events to a single exponential.³ From −5 to −20 mV HT blockade lifetimeswere measured by applying a cyclic sweep voltage protocol consisting of3 steps. In the first “capture” step, the applied potential was set to−60 mV for 2 seconds. In the second “release” step the applied potentialwas decreased to the voltage of interest (−5 to −20 mV) for 2-40 secwhere HT released from the pore. Finally in the “regeneration” step thepotential was briefly reversed to +35 mV for 0.2 seconds to regenerate anew unblocked open pore state. At least 50 sweeps were averaged and thepart of the trace corresponding to release step was fit to singleexponential. The duration of the FP blockades (dwell times), whichoccasionally showed both level 1 and level 2 currents, were distributedover two orders of magnitude and were not fit well with exponentialfunctions. Therefore, median dwell times are quoted for FP. The traceswere recorded at a sampling rate of 20 kHz with an internal low-passBessel filter set at 5 kHz. The measurements were performed in 150 mMNaCl, 15 mM Tris.HCl, pH 7.5. Graphs were made with Origin (OriginLabCorporation) and the temperature set at 28° C. All values quoted in thiswork are based on the average of at least three separate recordings,unless otherwise specified.

ClyA Pores for DNA Translocation DNA Preparation

ssDNA molecules were purchased from integrated DNA Technologies (IDT). 1was made by PCR where one of the two primers was 5′ biotinylated. 3 wasformed by incubating two complementary ssDNA molecules, one of whichcontained a biotin moiety at the 3′ end. The DNA hybrid was thenpurified from the excess ssDNA by affinity chromatography. 5 and 6 wereHPLC purified by IDT.

Preparation of ClyA Pores

ClyA was expressed in E. coli (DE3) pLysS cells by using a pT7 plasmid.Monomers containing a C-terminal oligo-histidine tag were expressed inE. coli cells and the soluble fraction purified by Ni-NTA affinitychromatography. ClyA dodecamers were formed by the addition of 0.2%β-dodecyl maltoside (DDM), and were separated from monomers by bluenative poly-acrylamide gel electrophoresis. The lowest band ofoligomeric ClyA-CS was extracted and stored at 4° C.

ClyA-2 nanopores were prepared by covalently attaching 2 to a ClyAprotein where the two WT cysteine residues (positions 87 and 285) weresubstituted with serine (ClyA-SS), and a cysteine was introduced atposition 103 (aspartate in the WT gene; ClyA-SSC₁₀₃). ClyA-SSC₁₀₃ wasconstructed from ClyA-SS, which also encoded a Gly-Ser-Ser linkerfollowed by a C-terminal hexahistidine tag, by using the megaprimermethod^(7a) using Phire® Hot Start DNA Polymerase (Finnzycnes). The DNA(5′-TTTTTTTTATCTACGAATTCATCAGGGCTAAAGAGTGCAGAGTTACTTAG-3′), containing aprotected thiol group attached to the 5′ hydroxyl of the DNA strand viaa C6 linker (5ThioMC6-D, IDT), was then conjugated to ClyA-SSC₁₀₃monomers, purified and oligomerised as described. Purified oligomerswere stored at −80° C. in 20% glycerol.

DNA Preparation

1 was made by PCR amplification of a pT7-ClyA-WT DNA template using a 5′biotinylated forward primer (bio-5′ TAATACGACTCACTATAGGG-3′) and anon-biotinylated reverse primer (5′-CATCAGCAGCACTTTGATATCGCCCACC-3′)using Taq DNA Polymerase from REDTaq® ReadyMix™ PCR Reaction Mix(Sigma). After a maximum number of 35 cycles the PCR product of 24reaction tubes (50 μL each tube) was purified by using a PCR quickpurification kit (QJAGEN) and the size of the construct checked by usinga 2% agarose gel (TAE buffer). The typical sample concentration was ˜200ng/μL.

3 was formed by incubating a 3′ biotinylated ssDNA molecule(5′-GGATGACCTGATCCAGATATTTATTATACAGGTCCAGCGCACCGTCAGCCCAATCGCACTTTTCACAAAAAGAGAGAGAGATCGATTACC-3′-bio, 3a) with a 20% excess of a partiallycomplimentary ssDNA(5′-GGTAATCGATCTCTCTCTCTTTTTGTGAAAAGTGCGATTGGGCTGACGGTGCGCTGGAC-3′, 3b,Table 4). The temperature was brought to 95° C. for one minute and thendecreased stepwise to room temperature. At around the estimatedannealing temperature, the temperature was decreased in 2° C. steps,each held for one minute. The hybrid DNA was then purified from theexcess of ssDNA by affinity chromatography, using a biotin-bindingcolumn containing monomeric avidin immobilised on agarose beads (ThermoScientific Pierce). 3 was eluted in Biotin Blocking/Elution Bufferaccording to the protocol. Typically a DNA concentration of ˜400 ng/μLwas obtained. The size of the dsDNA was checked by using a 2% agarosegel in TAE buffer. The purified dsDNA was stored at −20° C. in thepresence of 1 μM EDTA.

Electrical Recordings

If not otherwise specified, the signal was collected at sampling rate of50 KHz using a 10 kHz Bessel filter. The lipid bilayer was formed by theaddition of 1 to 2 μL of a 10% solution of1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in pentane (w/v).The electrical potential was applied by using Ag/AgCl electrodessubmerged in agar bridges (3% w/v low melt agarose in 2.5 M NaClbuffer). The applied potential refers to the potential of the workingelectrode connected to the trans compartment of the chamber. ClyAnanopore solutions (0.01-0.1 ng) were added to the cis compartment,which was connected to the ground electrode. After the insertion of asingle channel, excess protein was removed by several cycles ofperfusion. Electrical recordings were carried out in 2.5 M NaCl, 15 mMTris.HCl pH 8.0, at 22° C. The errors indicate the standard deviationfrom the average for at least three independent repeats, which areindicated with the letter n.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with theft full scope of equivalents, and the specification, alongwith such variations.

REFERENCES

-   -   1. (a) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D.        W., Characterization of individual polynucleotide molecules        using a membrane channel. Proc Natl Acad Sci U S A 1996, 93        (24), 13770-3; (b) Vercoutere, W.; Winters-Hilt, S.; Olsen, H.;        Deamer, D.; Haussler, D.; Akeson, M., Rapid discrimination among        individual DNA hairpin molecules at single-nucleotide resolution        using an ion channel. Nat Biotechnol 2001, 19 (3), 248.52; (c)        Howorka, S.; Cheley, S.; Bayley, H., Sequence-specific detection        of individual DNA strands using engineered nanopores. Nat        Biotechnol 2001, 19 (7), 636-9.    -   2. (a) Dekker, C., Solid-state nanopores. Nat Nanotechnol 2007,        2 (4), 209-15; (b) Li, J.; Stein, D.; McMullan, C.; Branton, D.;        Aziz, M. J.; Golovchenko, J. A., Ion-beam sculpting at nanometre        length scales. Nature 2001, 412 (6843), 166-9.    -   3. (a) Wei, R.; Martin, T. G.; Rant, U.; Dietz, H., DNA origami        gatekeepers for solid-state nanopores. Angew Chem Int Ed Engl        2012, 51 (20), 4864-7; (b) Bell, N. A.; Engst, C. R.; Ablay, M.;        Divitini, G.; Ducati, C.; Liedl, T.; Keyser, U. F., DNA origami        nanopores. Nano Lett 2012, 12 (1), 512-7; (c) Langecker, M.;        Arnaut, V.; Martin, T. G.; List, J.; Renner, S.; Mayer, M.;        Dietz, H.; Simmel, F. C., Synthetic lipid membrane channels        formed by designed DNA nanostructures. Science 2012, 338 (6109),        932-6.    -   4. Hall; Scott, A.; Rotem, D.; Mehta, K.; Bayley, H.; Dekker,        C., Hybrid pore formation by directed insertion of alpha        hemolysin into solid-state nanopores. Nature Nanotechnology        2011, In press.    -   5. Venkatesan, B. M.; Bashir, R., Nanopore sensors for nucleic        acid analysis. Nat Nanotechnol 2011, 6 (10), 615-24.    -   6. (a) Jung, Y.; Bayley, H.; Movileanu, L.,        Temperature-responsive protein pores. J Am Chem Soc 2006, 128        (47), 15332-40; (b) Heinz, C.; Engelhardt, H.; Niederweis, M.,        The core of the tetrameric mycobacterial porin MspA is an        extremely stable beta-sheet domain. J Biol Chem 2003, 278 (10),        8678-85.    -   7. (a) Soskine, M.; Biesemans, A.; Moeyaert, B.; Cheley, S.;        Bayley, H.; Maglia, G., An Engineered ClyA Nanopore Detects        Folded Target Proteins by Selective External Association and        Pore Entry. Nano Lett 2012, 12 (9), 4895-900; (b) Franceschini,        L.; Mikhailova, E.; Bayley, H.; Maglia, G., Nucleobase        recognition at alkaline pH and apparent pKa of single DNA bases        immobilised within a biological nanopore. Chem Commun (Camb)        2012, 48 (10), 1520-2; (c) Maglia, M.; Henricus, M.; Wyss, R.;        Li, Q.; Cheley, S.; Bayley, H., DNA strands from denatured        duplexes are translocated through engineered protein nanopores        at alkaline pH. Nano Letters 2009, 9, 3831-3836.    -   8. (a) Pastoriza-Gallego, M.; Oukhaled, G.; Mathe, J.; Thiebot,        B.; Betton, J. M.; Auvray, L. C.; Pelta, J., Urea denaturation        of alpha-hemolysin pore inserted in planar lipid bilayer        detected by single nanopore recording: Loss of structural        asymmetry. FEBS Lett 2007, 581 (18), 3371-3376; (b) Japrung, D.;        Henricus, M.; Li, Q. H.; Maglia, G.; Bayley, H., Urea        Facilitates the Translocation of Single-Stranded DNA and RNA        Through the alpha-Hemolysin Nanopore. Biophysical Journal 2010,        98 (9), 1856-1863.    -   9. (a) Mohammad, M. M.; Iyer, R.; Howard, K. R.; McPike, M. P.;        Borer, P. N.; Movileanu, L., Engineering a Rigid Protein Tunnel        for Biomolecular Detection. J Am Chem Soc 2012; (b) Bikwemu, R.;        Wolfe, A. J.; Xing, X.; Movileanu, L., Facilitated translocation        of polypeptides through a single nanopore. J Phys Condens Matter        2010, 22 (45), 454117; (c) Wolfe, A. J.; Mohammad, M. M.;        Cheley, S.; Bayley, H.; Movileanu, L., Catalyzing the        translocation of polypeptides through attractive interactions.        Journal of the American Chemical Society 2007, 129 (45),        14034-14041; (d) Movileanu, L.; Schmittschmitt, J. P.;        Scholtz, J. M.; Bayley, H., Interactions of peptides with a        protein pore. Biophys J 2005, 89 (2), 1030-45; (e) Payet, L.;        Martinho, M.; Pastoriza-Gallego, M.; Betton, J. M.; Auvray, L.;        Pelta, J.; Mathe, J., Thermal unfolding of proteins probed at        the single molecule level using nanopores. Anal Chem 2012, 84        (9), 4071-6; (f) Pastoriza-Gallego, M.; Rabah, L.; Gibrat, G.;        Thiebot, B.; van der Goot, F. G.; Auvray, L; Betton, J. M.;        Pelta, J., Dynamics of unfolded protein transport through an        aerolysin pore. J Am Chem Soc 2011, 133 (9), 2923-31; (g)        Oukhaled, G.; Mathé, J.; Biance, A.-L.; Bacri, L.; Betton,        J.-M.; Lairez, D.; Pelta, J.; Auvray, L., Unfolding of Proteins        and Long Transient Conformations Detected by Single Nanopore        Recording. Phys. Rev. Lett. 2007, 98, 158101; (h) Stefureac, R.        I.; Kachayev, A.; Lee, J. S., Modulation of the translocation of        peptides through nanopores by the application of an AC electric        field. Chem Commun (Camb) 2012, 48 (13), 1928-30; (i)        Stefureac, R. I.; Lee, J. S., Nanopore analysis of the folding        of zinc fingers. Small 2008, 4 (10), 1646-50; (j) Stefureac, R.;        Waldner, L.; Howard, P.; Lee, J. S., Nanopore analysis of a        small 86-residue protein. Small 2008, 4 (1), 59-63; (k)        Stefureac, R.; Long, Y. T.; Kraatz, H. B.; Howard, P.; Lee, J.        S., Transport of alpha-helical peptides through alpha-hemolysin        and aerolysin pores. Biochemistry 2006, 45 (30), 9172-9.    -   10. (a) Firnkes, M.; Pedone, D.; Knezevic, J.; Doblinger, M.;        Rant, U., Electrically Facilitated Translocations of Proteins        through Silicon Nitride Nanopores: Conjoint and Competitive        Action of Diffusion, Electrophoresis, and Electroosmosis. Nano        Letters 2010, 10 (6), 2162-2167; (b) Plesa, C. Kowalczyk, S. W.;        Zinsmeester, R.; Grosberg, A. Y.; Rabin, Y.; Dekker, C., Fast        Translocation of Proteins through Solid State Nanopores. Nano        Lett 2013; (c) Niedzwiecki, D. J.; Grazul, J.; Movileanu, L.,        Single-Molecule Observation of Protein Adsorption onto an        inorganic Surface. Journal of the American Chemical Society        2010, 132 (31), 10816-10822; (d) Fologea, D.; Ledden, B.;        McNabb, D. S.; Li, J. L., Electrical characterization of protein        molecules by a solid-state nanopore. Applied Physics Letters        2007, 91 (5); (e) Han, A.; Creus, M.; Schurmann, G.; Under, V.;        Ward, T. R.; de Rooij, N. F.; Staufer, U., Label-free detection        of single protein molecules and protein-protein interactions        using synthetic nanopores. Anal Chem 2008, 80 (12), 4651-8; (f)        Stefureac, R. I.; Trivedi, D.; Marziali, A.; Lee, J. S.,        Evidence that small proteins translocate through silicon nitride        pores in a folded conformation, J Phys Condens Matter 2010, 22        (45), 454133.    -   12. Eifler, N.; Vetsch, M.; Gregorini, M.; Ringler, P.; Chami,        M.; Philippsen, A.; Fritz, A.; Muller, S. A.; Glockshuber, R.;        Engel, A.; Grauschopf, U., Cytotoxin ClyA from Escherichia coli        assembles to a 13-meric pore independent of its redox-state.        EMBO J 2006, 25 (11), 2652-61.    -   13. (a) Pogoryelov, D.; Klyszejko, A. L.; Krasnoselska, G. O.;        Heller, E. M.; Leone, V.; Langer, J. D.; Vonck, J.; Muller, D.        J.; F-araldo-Gomez, J. D.; Meier, T., Engineering rotor ring        stolchlometries in the ATP synthase. Proc Natl Acad Sci U S A        2012, 109 (25), E1599-608; (b) Bayfield, O. W.; Chen, C. S.;        Patterson, A. R.; Luan, W.; Smits, C.; Gollnick, P.; Antson, A.        A., Trp RNA-binding attenuation protein: modifying symmetry and        stability of a circular oligomer. PLoS One 2012, 7 (9), e44309.    -   14. Niedzwiecki, D. J.; Iyer, R.; Borer, P. N.; Movileanu, L.,        Sampling a Biomarker of the Human Immunodeficiency Virus across        a Synthetic Nanopore. ACS Nano 2013.    -   15. (a) Clarke, J.; Wu, H.; Jayasinghe, L.; Patel, A.; Reid, S.;        Bayley, H., Continuous base identification for single-molecule        nanopore DNA sequencing. Nature Nanotechnology 2009, 4,        265-270; (b) Rincon-Restrepo, M.; Mikhailova, E.; Bayley, H.;        Maglia, G., Controlled Translocation of Individual DNA Molecules        through Protein Nanopores with Engineered Molecular Brakes. Nano        Lett 2011, 11 (2), 746-50.    -   16. (a) Freedman, K. J.; Jurgens, M.; Prabhu, A.; Ahn, C. W.;        Jemth, P.; Edel, J. B.; Kim, M. J., Chemical, thermal, and        electric field induced unfolding of single protein molecules        studied using nanopores. Anal Chem 2011, 83 (13), 5137-44; (b)        Niedzwiecki, D. J.; Movileanu, L., Monitoring protein adsorption        with solid-state nanopores. J Vis Exp 2011, (58); (c) Talaga, D.        S.; Li, J., Single-molecule protein unfolding in solid state        nanopores. J Am Chem Soc 2009, 131 (26), 9287-97.    -   17. Mueller, M.; Grauschopf, U.; Maier, T.; Glockshuber, R.;        Ban, N., The structure of a cytolytic alpha-helical toxin pore        reveals its assembly mechanism. Nature 2009, 459 (7247),        726-U135.    -   18. Maglia, G.; Heron, A. J.; Stoddart, D.; Japrung, D.; Bayley,        H., Analysis of Single Nucleic Acid Molecules with Protein        Nanopores. Methods in Enzymology Vol 475: Single Molecule Tools,        Pt B 2010, 474, 591-623.    -   19, Miyazaki, K., MEGAWHOP cloning: a method of creating random        mutagenesis libraries via megaprimer PCR of whole plasmids.        Methods Enzymol 2011, 498, 399-406.    -   20. Delhaise, P.; Bardiaux, M.; Demaeyer, M.; Prevost, M.;        Vanbelle, D.; Donneux, J.; Lasters, I.; Vancustem, E.; Alard,        P.; Wodak, S. J., The Brugel Package—toward        Computer-Aided-Design of Macromolecules. J Mol Graphics 1988, 6        (4), 219-219.    -   21. King, N. P. et al. Computational design of self-assembling        protein nanomaterials with atomic level accuracy. Science 336,        1171-1174 (2012).    -   22. Ackermann, D. et al. A double-stranded DNA rotaxane. Nat        Nanotechnol 5, 436-442 (2010).    -   23. Sanchez-Quesada, J., Saghateiian, A., Cheley, S., Bayley, H.        & Ghadiri, M. R. Single DNA rotaxanes of a transmembrane pore        protein. Angew Chem Int Ed Engl 43, 3063-3067 (2004).    -   24. Meller, A., Nivon, L. & Branton, D. Voltage-driven DNA        translocations through a nanopore. Phys Rev Lett 86, 3435-3438        (2001).    -   25. Henrickson, S. E., Misakian, M., Robertson, B. &        Kasianowicz, J. J. Driven DNA transport into an asymmetric        nanometer-scale pore. Phys Rev Lett 85, 3057-3060 (2000).    -   26. Maglia, G., Rincon Restrepo, M., Mikhailova, E. & Bayley, H.        Enhanced translocation of single DNA molecules through        α-hemolysin nanopores by manipulation of internal charge. Proc        Natl Acad Sci U S A 105, 19720-19725 (2008).    -   27. Butler, T. Z., Pavlenok, M., Derrington, I. M.,        Niederweis, M. & Gundlach, J. H. Single-molecule DNA detection        with an engineered MspA protein nanopore. Proc Natl Acad Sci U S        A 105, 20647-20652 (2008).    -   28. Wanunu, M., Morrison, W., Rabin, Y., Grosberg, A. Y. &        Meller, A. Electrostatic focusing of unlabelled DNA into        nanoscale pores using a salt gradient. Nat Nanotechnol 5,        160-165 (2010).

TABLES

TABLE 1 Parameters for ClyA-SS and the three types of ClyA-CS nanopores.The diameter of Type I ClyA-SS and ClyA-CS was taken from the crystalstructure of E. coli ClyA. The diameter of Type II and Type III ClyA-CSwas measured from models that were created by adding one (Type II) ortwo (Type III) subunits to the structure of the 12mer ClyA (crystalstructure) as described in the supplementary information. The diametersof the nanopores were determined including the Van der Waals radii ofthe atoms (supplementary information). Errors are given as standarddeviations. Type I* Type I ClyA- Type II Type III Parameters ClyA-SS CSClyA-CS ClyA-CS Trans diameter, nm 3.3 3.3 3.7 4.2 Cis diameter, nm 5.55.5 5.9 6.5 Nanopore conductance at −35 mV, nS  1.8 ± 0.1 1.78 ± 0.04 2.19 ± 0.09 2.81 ± 0.11 Nanopore conductance at −150 mV, nS NA 1.50 ±0.03  1.85 ± 0.06 NA HT Occupancy of L2 at −35 mV, % 22 ± 5 30 ± 10 96 ±2 100    HT Level 1 at −35 mV, I_(RES) % 56 ± 1 56 ± 1  68 ± 1 NA HTLevel 2 at −35 mV, I_(RES) % 23 ± 1 23 ± 3  31 ± 1 32 ± 9  HT Occupancyof L2 at −150 mV, % NA 100    100    NA HT Level 2 (−150 mV), I_(RES), %NA 23 ± 2  31 ± 5 NA HT Dwell time at −150 mV, ms NA 1.0 ± 0.4  235 ±186 NA (*Data taken from^(7a))

TABLE 2 Mutations accumulated during the directed evolution rounds ofthe ClyA-SS gene. Round Name Clone ID Sequence changes relative toWT-ClyA 0 ClyA-SS dSClyA C87S, C285S 3 3ClyA1 C87S, F166Y, K230R, C285S3 3ClyA2 Q73R, F166Y, C285S 3 3ClyA3 Q33R, Q56H, C87S, D122G, C285S 44ClyA1 I4T, N128S, S145I, C285S 4 4ClyA2 S110I, C285S, F166Y, T223A 44ClyA3 T39I, C285S, F166Y, K230R 4 ClyA-CS 4ClyA4 L99Q, E103G, F166Y,C285S, K294R 4 4ClyA5 I4T, Q73R, C285S 4 4ClyA6 Q73R, F166Y, C285S 55ClyA1 C87A, L99Q, E103G, C285S, F166Y, N220S, Q289R, K294R, H307Y 55ClyA2 C87A, L99Q, E103G, C285S, F166Y, Q289R, K294R, H307Y 5 ClyA-AS5ClyA3 C87A, L99Q, E103G, C285S, F166Y, I203V, K294R, H307Y

TABLE 3 Primers: N stands for A, G, C, or T; S is G or C, thus NNS codon encodes for the full set of amino adds. SEQ ID NameSequence  NO 87NNS GAAGCTACCCAAACGGTTTACGAATG 17GNNSGGTGTGGTTACCCAGCTGCTG T7 promoter TAATACGACTCACTATAGGG 18 T7GCTAGTTATTGCTCAGCGG 19 terminator 87C GTTTACGAATGGTGTGGTGTGGTTACCCAG 20285C CGCTGCTGATATTCATTACAGGTATTAAT 21 CATTTTC

TABLE 4 Summary of DNA molecules used in this work.1 was prepared by PCR as described in methods. 3 was formed by incubating 3a with a 20% excess of 3b and purified by affinity chromatogtraphy as described in  methods. The complimentary sequences in the two DNA strandsare shown in italics. SEQ ID Name DNA sequence NO 1 1a Bio- 75′TAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGACGGGTATCTTTGCGGAACAGACGGTGGAAGTTGTGAAAAGTGCGATTGAAACGGCTGACGGTGCGCTGGACCTGTATAATAAATATCTGGATCAGGTCATCCCGTGGAAAACCTTTGACGAAACGATTAAAGAACTGAGCCGTTTCAAACAGGAATACAGTCAAGAAGCGTCCGTCCTGGTGGGCGATATCAAAGTGCTGCTGATG3′ 1b5′CATCAGCAGCACTTTGATATCGCCCACCAGGACGGACGCTTCTTGACTGTAT 8TCCTGTTTGAAACGGCTCAGTTCTTTAATCGTTTCGTCAAAGGTTTTCCACGGGATGACCTGATCCAGATATTTATTATACAGGTCCAGCGCACCGTCAGCCGTTTCAATCGCACTTTTCACAACTTCCACCGTCTGTTCCGCAAAGATACCCGTCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGAAACCGTTGTGGTCTCCCTATAGTGAGTCGTATTA3′ 25′TTTTTTTTTATCTACGAATTCATCAGGGCTAAAGAGTGCAGAGTTACTTAG3′ 9 3 3a5′GGATGACCTGATCCAGATATTTATTATACAGGTCCAGCGCACCGTCAGCCCA 10ATCGCACTTTTCACAAAAAGAGAGAGAGATCGATTACC3′-bio 3b5′GGTAATCGATCTCTCTCTCTTTTTGTGAAAAGTGCGATTGGGCTGACGGTGC 11 GCTGGAC-3′ 45′AATAAATATCTGGATCAGGTCATCCCTAAGTAACTCTGCAC3′ 12 5a5′GGATGACCTGATCCAGATATTTATTATACAGGTCCAGCGCACCGTCAGCCCA 13ATCGCACTTTTCACAAAAAGAGAGAGAGATCGATTACC3′bio 5b5′GGTAATCGATCTCTCTCTCTTTTTGTGAAAAGTGCGATTGGGCTGACGGTGC 14GCTGGACCTGTATAATAAATATCTGGATCAGGTCATCC3′ 65′GCCCTATATTATCAGGTCATCCCTAAGTAACTCTGCA3′ 15 75′GCCCTATATTATCAGGTCATCCCTAAGTAACTCTGCA3′ 16

ClyA SEQUENCES Description Sequence SEQ ID NO Protein sequence for S.MIMTGIFAEQTVEVVKSAIETADGALDLYNKYLDQVIPWKTFDETIKEL SEQ ID NO: 1typhi ClyA (ClyA-WT) SRFKQEYSQEASVLVGDIKVLLIMDSQDKYFEATQTVYEWCGVVTQLLSAYILLFDEYNEKKASAQKDILIRILDDGVKKLNEAQKSLLTSSQSFNNASGKLLALDSQLTNDFSEKSSYFQSQVDRIRKEAYAGAAAGIVAGPFGLIISYSIAAGVIEGKLIPELNNRLKTVQNFFTSLSATVKQANKDIDAAKLKLATEIAAIGEIKTETETTRFYVDYDDLMLSLLKGAAKKMINTCNEYQQRHGKKTL FEVPDVProtein sequence for MTGIFAEQTVEVVKSAIETADGALDLYNKYLDQVIPWKTFDETIKELSRFSEQ ID NO: 2 ClyA with C285SKQEYSQEASVLVGDIKVLLMIDSQDKYFEATQTVYEWCGVVTQLLSAYI substitution (ClyA-CS)QLFDGYNEKKASAQKDILIRILDDGVKKLNEAQKSLLTSSQSFNNASGKLLALDSQLTNDFSEKSSYYQSQVDRIRKEAYAGAAAGIVAGPFGLIISYSIAAGVIEGKLIPELNNRLKTVQNFFTSLSATVKQANKDIDAAKLKLATEIAAIGEIKTETETTRFYVDYDDLMLSLLKGAAKKMINTSNEYQQRHGRKTLFE VPDVGSSHHHHHH*Protein sequence for MTGIFAEQTVEVVKSAIETADGALDLYNKYLDQVIPWKTFDETIKELSRFSEQ ID NO: 3 ClyA-AS KQEYSQEASVLVGDIKVLLMDSQDKYFEATQTVYEWAGVVTQLLSAYIQLEDGYNEKKASAQKDILIRILDDGVKKLNEAQKSLLTSSQSFNNASGKLLALDSQLTNDFSEKSSYYQSQVDRIRKEAYAGAAAGIVAGPFGLIISYSIAAGVVEGKLIPELNNRLKTVQNFFTSLSATVKQANKDIDAAKLKLATEIAAIGEIKTETETTRFYVDYDDLMLSLLKGAAKKMINTSNEYQQRHGRKTLF EVPDVGSSYHHHHH*Nucleotide sequence for CCTGCGTAGATAAGCAGGAAGCAGGCAGTATTTCCAGCTTCTGGAASEQ ID NO: 4 S. typhi ClyATGTTAAAGCTACAAAAGTTGTCTGGAGGTAATAGGTAAGAATACTTTATAAAACAGGTACTTAATTGCAATTTATATATTTAAAGAGGCAAATGATTATGACCGGAATATTTGCAGAACAAACTGTAGAGGTAGTTAAAAGCGCGATCGAAACCGCAGATGGGGCATTAGATCTTTATAACAAATACCTCGACCAGGTCATCCCCTGGAAGACCTTTGATGAAACCATAAAAGAGTTAAGCCGTTTTAAACAGGAGTACTCGCAGGAAGCTTCTGTTTTAGTTGGTGATATTAAAGTTTTGCTTATGGACAGCCAGGACAAGTATTTTGAAGCGACACAAACTGTTTATGAATGGTGTGGTGTCGTGACGCAATTACTCTCAGCGTATATTTTACTATTTGATGAATATAATGAGAAAAAAGCATCAGCCCAGAAAGACATTCTCATTAGGATATTAGATGATGGTGTCAAGAAACTGAATGAAGCGCAAAAATCTCTCCTGACAAGTTCACAAAGTTTCAACAACGCTTCCGGAAAACTGCTGGCATTAGATAGCCAGTTAACTAATGATTTTTCGGAAAAAAGTAGTTATTTCCAGTCACAGGTGGATAGAATTCGTAAGGAAGCTTATGCCGGTGCTGCAGCCGGCATAGTCGCCGGTCCGTTTGGATTAATTATTTCCTATTCTATTGCTGCGGGCGTGATTGAAGGGAAATTGATTCCAGAATTGAATAACAGGCTAAAAACAGTGCAAAATTTCTTTACTAGCTTATCAGGTACAGTGAAACAAGCGAATAAAGATATCGATGCGGCAAAATTGAAATTAGCCACTGAAATAGCAGCAATTGGGGAGATAAAAACGGAAACCGAAACAACCAGATTCTACGTTGATTATGATGATTTAATGCTTTCTTTATTAAAAGGAGCTGCAAAGAAAATGATTAACACCTGTAATGAATACCAACAAAGACACGGTAAGAAGACGCTTTTCGAGGTTCCTGACGTCTGATACATTTTCATTCGATCTGTGTACTTTTAACGCCCGATAGCGTAAAGAAAATGAGAGACGGAGAAAAAGCGATATTCAACAGCCCGATAAACAAGAGTCGTTACCGGGCTGACGAGGTTATCAGGCGTTAAGCTGGTAG Nucleotide sequence forATGACGGGTATCTTTGCGGAACAGACGGTGGAAGTTGTGAAAAGT SEQ ID NO: 5ClyA with C285S GCGATTGAAACGGCTGACGGTGCGCTGGACCTGTATAATAAATATCsubstitution (ClyA-CS) TGGATCAGGTCATCCCGTGGAAAACCTTTGACGAAACGATTAAAGAACTGAGCCGTTTCAAACAGGAATACAGTCAAGAAGCGTCCGTCCTGGTGGGCGATATCAAAGTGCTGCTGATGGATTCTCAGGACAAATATTTTGAAGCTACCCAAACCGGTTTACGAATGGTGTGGTGTGGTTACCCAGCTGCTGTCCGCATATATTCAGCTGTTCGATGGATACAACGAGAAAAAAGCGAGCGCGCAGAAAGACATTCTGATCCGCATTCTGGATGACGGCGTGAAAAAACTGAATGAAGCCCAGAAATCGCTGCTGACCAGCTCTCAATCATTTAACAATGCCTCGGGTAAACTGCTGGCACTGGATAGCCAGCTGACGAACGACTTTTCTGAAAAAAGTTCCTATTACCAGAGCCAAGTCGATCGTATTCGTAAAGAAGCCTACGCAGGTGCCGCAGCAGGTATTGTGGCCGGTCCGTTCGGTCTGATTATCTCATATTCGATTGCTGCGGGCGTTATCGAAGGTAAACTGATTCCGGAACTGAACAATCGTCTGAAAACCGTTCAGAACTTTTTCACCAGTCTGTCTGCTACGGTCAAACAAGCGAATAAAGATATCGACGCCGCAAAACTGAAACTGGCCACGGAAATCGCTGCGATTGGCGAAATCAAAACCGAAACGGAAACCACGCGCTTTTATGTTGATTACGATGACCTGATGCTGAGCCTGCTGAAAGGTGCCGCGAAGAAAATGATTAATACCTCTAATGAATATCAGCAGCGTCACGGTAGAAAAACCCTGTTTGAAGTCCCGGATGTGGGCAGCAGCCACCACCATCATCACCACTAAAAGCTTGGATCCGGCTGCTAACAAAGCCC GAANucleotide sequence for ATGACGGGTATCTTTGCGGAACAGACGGTGGAAGTTGTGAAAAGTSEQ ID NO: 6 ClyA-AS GCGATTGAAACGGCTGACGGTGCGCTGGACCTGTATAATAAATATCTGGATCAGGTCATCCCGTGGAAAACCTTTGACGAAACGATTAAAGAACTGAGCCGTTTCAAACAGGAATACAGTCAAGAAGCGTCCGTCCTAGTGGGCGATATCAAAGTGCTGCTGATGGATTCTCAGGACAAATATTTTGAAGCTACCCAAACGGTTTACGAATGGGCGGGTGTGGTTACCCAGCTGCTGTCCGCATATATTCAGCTGTTCGATGGATACAATGAGAAAAAAGCGAGCGCGCAGAAAGACATTCTGATCCGCATTCTGGATGACGGCGTGAAAAAACTGAATGAAGCCCAGAAATCGCTGCTGACCAGCTCTCAATCATTTAACAATGCCTCGGGTAAACTGCTGGCACTGGATAGCCAGCTGACGAACGACTTTTCTGAAAAAAGTTCCTATTACCAGAGCCAAGTCGATCGTATTCGTAAAGAAGCCTACGCAGGTGCCGCAGCAGGTATTGTGGCCGGTCCGTTCGGTCTGATTATCTCATATTCAATTGCTGCGGGCGTTGTCGAAGGTAAACTGATTCCGGAACTGAACAATCGTCTGAAAACCGTTCAGAACTTTTTCACCAGTCTGTCTGCTACGGTCAAACAAGCGAATAAAGATATCGACGCCGCAAAACTGAAACTGGCCACGGAAATCGCTGCGATTGGCGAAATCAAAACCGAAACGGAAACCACGCGCTTTTATGTTGATTACGATGACCTGATGCTGAGCCTGCTGAAAGGTGCCGCGAAGAAAATGATTAATACCTCTAATGAATATCAGCAGCGTCACGGTAGAAAAACCCTGTTTGAAGTCCCGGATGTGGGCAGCAGCTAC CACCATCATCACCACTAAAAGCTT

1-16. (canceled)
 17. A nanopore sensor system comprising: i) afluid-filled compartment separated by a membrane into a first chamberand a second chamber, wherein the fluid is an ionic solution; ii) a ClyApore inserted in the membrane, wherein the ClyA pore comprises 14subunits; and iii) electrodes configured for generating an electricalpotential difference across the membrane to facilitate ionic flowthrough the ClyA pore from the first chamber to the second chamber. 18.The nanopore sensor system of claim 17 further comprising a ligand incombination with the ClyA pore.
 19. The nanopore sensor system of claim17, wherein the ClyA pore comprises a plurality of subunits, eachsubunit comprising a polypeptide represented by an amino acid sequencewith at least 80% identity to SEQ ID NO:1; SEQ ID NO:2; or SEQ ID NO:3.20. The nanopore sensor according to claim 18, wherein the ligandconfers selective binding properties to an analyte present in the ionicsolution.
 21. The nanopore sensor according to claim 20, wherein theligand is an aptamer.
 22. The nanopore sensor according to claim 20,wherein the ligand is selected to bind to a specific target proteinanalyte.
 23. The nanopore sensor according to claim 20, wherein theligand is an antibody.
 24. The nanopore sensor according to claim 20,wherein the ligand is a receptor.
 25. The nanopore sensor according toclaim 20, wherein the ligand is a peptide.
 26. The nanopore sensoraccording to claim 22, wherein the protein analyte binds within thelumen of the ClyA pore.
 27. The nanopore sensor according to claim 22,wherein the protein analyte is a protein with a molecular weight in therange of 15-70 kDa.
 28. The nanopore sensor according to claim 26,wherein the protein analyte is a protein with a molecular weight in therange of 15-70 kDa.
 29. The nanopore sensor according to claim 17,wherein the lumen of the ClyA pore is modified to alter the size,binding properties, and/or structure of the pore.
 30. The nanoporesensor according to claim 18, wherein the ClyA pore is attached to theligand.
 31. The nanopore sensor according to claim 30, wherein the ClyApore is attached to the ligand via a disulfide linkage.
 32. The nanoporesensor according to claim 30, wherein the ClyA pore is attached to theligand via cross-linking.
 33. The nanopore sensor according to claim 30,wherein the ClyA pore is attached to the ligand via chemical ligation.34. The nanopore sensor according to claim 18, wherein the ClyA pore isnot attached to the ligand.
 35. A method for detecting a target analytein a sample comprising: (a) contacting the sample with the nanoporesensor system of claim 1; (b) applying one or more electric potentialsacross the ClyA pore of the nanopore sensor system; (c) measuringcurrent passing through the ClyA pore at each of the one or moreelectrical potentials; and (d) comparing measured currents withreference currents, wherein a change in current relative to thereference currents indicates that the target analyte is present in thesample.